Methods and Apparatuses for Chip-Based DNA Error Reduction

ABSTRACT

Methods and apparatus relate to reduction of sequence errors generated during synthesis of nucleic acids on a microarray chip. The error reduction can include synthesis of complementary stands (to template strands), using a short universal primer complementary to the template strands and polymerase. Heteroduplex can be formed be melting and re-annealing complementary stands and template strands. The heteroduplexes containing a mismatch can be recognized and cleaved by a mismatch endonuclease. The mismatch-containing cleaved heteroduplexes can be removed from the microarray chip using a global buffer exchange. The error free synthetic nucleic acids generated therefrom can be used for a variety of applications, including synthesis of biofuels and value-added pharmaceutical products.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/164,045, filed Jun. 20, 2011, which is a continuation ofInternational Application No. PCT/US2010/057405, filed Nov. 19, 2010,now expired, which claims the benefit of and priority to U.S.Provisional Patent Application No. 61/264,643, filed Nov. 25, 2009, nowexpired, the entire contents of each of which applications are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

Methods and apparatuses provided herein relate to the synthesis andassembly of high fidelity nucleic acids and nucleic acid librarieshaving a predefined sequence using microvolume reactions. Moreparticularly, methods and apparatuses are provided for polynucleotidesynthesis, error reduction, hierarchical assembly, and/or sequenceverification on a solid support. In some embodiments, pico-liter and subpico-liter dispensing and droplet moving technologies are applied toaccess and manipulate the oligonucleotides on DNA microarrays.

BACKGROUND

Using the techniques of recombinant DNA chemistry, it is now common forDNA sequences to be replicated and amplified from nature and thendisassembled into component parts. As component parts, the sequences arethen recombined or reassembled into new DNA sequences. However, relianceon naturally available sequences significantly limits the possibilitiesthat may be explored by researchers. While it is now possible for shortDNA sequences to be directly synthesized from individual nucleosides, ithas been generally impractical to directly construct large segments orassemblies of polynucleotides, i.e., polynucleotide sequences longerthan about 400 base pairs.

Oligonucleotide synthesis can be performed through massively parallelcustom syntheses on microchips (Zhou et al. (2004) Nucleic Acids Res.32:5409; Fodor et al. (1991) Science 251:767). However, currentmicrochips have very low surface areas and hence only small amounts ofoligonucleotides can be produced. When released into solution, theoligonucleotides are present at picomolar or lower concentrations persequence, concentrations that are insufficiently high to drivebiomolecular priming reactions efficiently. Current methods forassembling small numbers of variant nucleic acids cannot be scaled up ina cost-effective manner to generate large numbers of specified variants.As such, a need remains for improved methods and devices forhigh-fidelity gene assembly and the like.

Furthermore, oligonucleotides on microchips are generally synthesizedvia chemical reactions. Spurious chemical reactions cause random baseerrors in oligonucleotides. One of the critical limitations in chemicalnucleic acid synthesis is the error-rate. The error rate ofchemically-synthesized oligonucleotides (deletions at a rate of 1 in 100bases and mismatches and insertions at about 1 in 400 bases) exceeds theerror rate obtainable through enzymatic means of replicating an existingnucleic acid (e.g., PCR). Therefore, there is an urgent need for newtechnology to produce high-fidelity polynucleotides.

SUMMARY

Aspects of the invention relate to methods and apparatuses for preparingand/or assembling high fidelity polymers. Also provided herein aredevices and methods for processing nucleic acid assembly reactions andassembling nucleic acids. It is an object of this invention to providepractical, economical methods of synthesizing custom polynucleotides. Itis a further object to provide a method of producing syntheticpolynucleotides that have lower error rates than syntheticpolynucleotides made by methods known in the art.

According to one embodiment, the invention provides a method forproducing high fidelity oligonucleotides on a solid support, comprisingthe steps of exposing a plurality of support-bound single-strandedoligonucleotides comprising a predefined sequence to a polymerase enzymeunder conditions suitable for a template-dependent synthesis reaction,thereby to produce a plurality of double-stranded oligonucleotides, eachof which comprises a support-bound oligonucleotide and a synthesizedcomplementary oligonucleotide; denaturing the plurality ofdouble-stranded oligonucleotides such that the synthesizedoligonucleotides are released from the support-bound oligonucleotidesinto a solution; reannealing the synthesized oligonucleotides to thesupport-bound oligonucleotides, thereby to produce reannealeddouble-stranded oligonucleotides; and exposing the reannealeddouble-stranded oligonucleotides to a mismatch recognizing and cleavingcomponent under conditions suitable for cleavage of reannealeddouble-stranded oligonucleotides containing a mismatch.

In one aspect, the invention relates to a method for producing highfidelity oligonucleotides on a solid support. The method includessynthesizing a first plurality of oligonucleotides in a chain extensionreaction using a second plurality of oligonucleotides as templates. Thesecond plurality of oligonucleotides are immobilized on a solid supportand comprises an error-containing oligonucleotide having a sequenceerror at an error-containing position. The chain extension reactionproduces a first plurality of duplexes. The method also includes meltingthe first plurality of duplexes to release the first plurality ofoligonucleotides from the second plurality of oligonucleotides, whereinthe first plurality of oligonucleotides comprise error-freeoligonucleotides that are free of error at the error-containing positionof the error-containing oligonucleotide. The method further includescontacting the first plurality of oligonucleotides with the secondplurality of oligonucleotides under hybridization conditions to form asecond plurality of duplexes. The second plurality of duplexes comprisesa mismatch-containing heteroduplex formed between the error-containingoligonucleotide and one of the error-free oligonucleotides. The methodalso includes removing at least a portion of the mismatch-containingheteroduplex by a mismatch recognizing and cleaving component, therebyproducing high fidelity oligonucleotides. In some embodiments, themethod further includes after the removing step, selectively meltingaway truncations.

In another aspect, the invention relates to a method of assemblingnucleic acid polymers. The method includes producing two or more poolsof high fidelity oligonucleotides according to the methods describedherein. The method also include melting desirable pools of high fidelityoligonucleotides into a solution, combining the desirable pools of highfidelity oligonucleotides into a reaction volume, and subjecting thereaction volume to conditions suitable for one or more of hybridization,ligation, and/or chain extension.

In various embodiments, the second plurality of oligonucleotides arechemically synthesized on the solid support and immobilized within oneor more features on the solid support. The second plurality ofoligonucleotides can include oligonucleotides having substantially thesame sequence. The second plurality of oligonucleotides can be depositedat one feature on the solid support, or at two or more features on thesolid support. The second plurality of oligonucleotides can be of two ormore different sequences and each sequence is found at a differentfeature on the solid support. In some embodiments, the solid support isa microarray.

In some embodiments, the first plurality of oligonucleotides isenzymatically synthesized on the solid support. One or more of the firstplurality of oligonucleotides can diffuse in a fluid when melted off andnot duplexed with the second plurality of oligonucleotides.

Some aspects of the invention relate to a method of producing at leastone oligonucleotide having a predefined sequence on a solid support, themethod comprising (a) synthesizing on a solid support a first pluralityof double-stranded oligonucleotides using a second plurality ofoligonucleotides as templates; (b) releasing the first plurality ofoligonucleotides from the second plurality of oligonucleotides within anisolated microvolume; (c) contacting the second plurality ofoligonucleotides with the first plurality of oligonucleotides underhybridization conditions to form a second plurality of double-strandedoligonucleotides within the isolated volume; (d) contacting and cleavingthe second plurality of double-stranded oligonucleotides with a mismatchbinding agent, wherein the mismatch binding agent selectively binds andcleaves the double-stranded oligonucleotides comprising a mismatch; and(e) removing the double-stranded oligonucleotides comprising themismatch thereby producing error-free oligonucleotides. In someembodiments, the first plurality of oligonucleotides is released underdenaturing conditions. In some embodiments, the method further comprisesreleasing error-free oligonucleotides in solution.

In some embodiments, the second plurality of oligonucleotides is boundto a discrete feature of the solid support and the feature isselectively hydrated thereby providing the second plurality ofoligonucleotide within the isolated volume. The feature can beselectively hydrated by spotting a solution comprising a polymerase,dNTPs, a solution promoting primer extension, at least one primerwherein the primer is complementary to a primer binding site on thesecond plurality of oligonucleotides.

In certain embodiments, the mismatch recognizing and cleaving componentcomprises a mismatch endonuclease. In one embodiment, the mismatchendonuclease is CEL1. In some embodiments, the mismatch recognizing andcleaving component performs chemical cleavage. After cleaving, certainembodiments can also include removing the cleaved heteroduplex havingthe mismatch by buffer exchange.

Other features and advantages of the devices and methods provided hereinwill be apparent from the following detailed description, and from theclaims. The claims provided below are hereby incorporated into thissection by reference. The various embodiments described herein can becomplimentary and can be combined or used together in a mannerunderstood by the skilled person in view of the teachings containedherein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an exemplary method of producing high fidelityoligonucleotides on a DNA microarray where heteroduplex formation isfollowed by mismatch cleavage.

FIG. 2 shows an exemplary Surveyor cleavage experiment of heteroduplexesafter shuffling.

FIG. 3 shows an exemplary graph showing Microarray Spot Intensity(signal strength) after Surveyor™ treatment.

FIG. 4 illustrates an exemplary method for producing high fidelityoligonucleotides using Surveyor cleavage: molecular reaction flow,process flow, and reagent flow.

FIG. 5A illustrates micro-volumes (502) suitable for shuffling andcovered by a blanket of fluid (503). FIG. 5B illustrates a process forthe creation of micro-volumes in a serial manner according to oneembodiment. FIG. 5C illustrates a process for the creation ofmicro-volumes according to another embodiment. FIG. 5C1 shows asubstrate fully immersed in a fluid (511). FIG. 5C2 shows the resultingmicro-volumes. FIG. 5D illustrates a process for the creation ofmicro-volumes according to another embodiment.

FIG. 6A1 shows the setting prior to melting with surface-attachedhomoduplexes. FIG. 6A2 shows the concentration of duplexes at twoadjacent positions on the substrate. FIG. 6B1 shows the melting at atime immediately after the temperature increases. FIG. 6B2 shows theconcentration of duplexes at two adjacent positions on the substrate.FIG. 6E shows the result after reannealing of the molecules according toone embodiment. FIG. 6C1 shows the melting at a later time than that ofFIG. 6B1. FIG. 6C2 shows the concentration of duplexes at two adjacentpositions on the substrate. FIG. 6F shows the result after reannealingof the molecules according to one embodiment. FIG. 6D1 shows the meltingat a later time than that of FIG. 6C1. FIG. 6D2 shows the concentrationof duplexes at two adjacent positions on the substrate. FIG. 6G showsthe result after reannealing of the molecules according to oneembodiment.

FIG. 7A illustrates homoduplexes formed by the template (702) and copies(703) on a surface (701). FIG. 7B illustrates the homoduplexes coveredby a volume (704). FIG. 7C illustrates the melted copies (706) in thefluid (705) which is reduced in volume. FIG. 7D illustrates anembodiment in which the droplet is allowed to completely dry, depositingthe copies (707) on the surface. FIG. 7E shows the substrate afterrehydration to resuspend the copies (707) into the fluid volume (708).

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the technology provided herein are useful for increasing theaccuracy, yield, throughput, and/or cost efficiency of nucleic acidsynthesis and assembly reactions. As used herein the terms “nucleicacid”, “polynucleotide”, “oligonucleotide” are used interchangeably andrefer to naturally-occurring or synthetic polymeric forms ofnucleotides. The oligonucleotides and nucleic acid molecules of thepresent invention may be formed from naturally occurring nucleotides,for example forming deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) molecules. Alternatively, the naturally occurring oligonucleotidesmay include structural modifications to alter their properties, such asin peptide nucleic acids (PNA) or in locked nucleic acids (LNA). Thesolid phase synthesis of oligonucleotides and nucleic acid moleculeswith naturally occurring or artificial bases is well known in the art.The terms should be understood to include equivalents, analogs of eitherRNA or DNA made from nucleotide analogs and as applicable to theembodiment being described, single-stranded or double-strandedpolynucleotides. Nucleotides useful in the invention include, forexample, naturally-occurring nucleotides (for example, ribonucleotidesor deoxyribonucleotides), or natural or synthetic modifications ofnucleotides, or artificial bases. As used herein, the term monomerrefers to a member of a set of small molecules which are and can bejoined together to from an oligomer, a polymer or a compound composed oftwo or more members. The particular ordering of monomers within apolymer is referred to herein as the “sequence” of the polymer. The setof monomers includes but is not limited to example, the set of commonL-amino acids, the set of D-amino acids, the set of synthetic and/ornatural amino acids, the set of nucleotides and the set of pentoses andhexoses. Aspects of the invention described herein primarily with regardto the preparation of oligonucleotides, but could readily be applied inthe preparation of other polymers such as peptides or polypeptides,polysaccharides, phospholipids, heteropolymers, polyesters,polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylenesulfides, polysiloxanes, polyimides, polyacetates, or any otherpolymers.

As used herein, the term “predetermined sequence” or “predefined” meansthat the sequence of the polymer is known and chosen before synthesis orassembly of the polymer. In particular, aspects of the invention aredescribed herein primarily with regard to the preparation of nucleicacids molecules, the sequence of the oligonucleotide or polynucleotidebeing known and chosen before the synthesis or assembly of the nucleicacid molecules. In some embodiments of the technology provided herein,immobilized oligonucleotides or polynucleotides are used as a source ofmaterial. In various embodiments, the methods described herein useoligonucleotides, their sequences being determined based on the sequenceof the final polynucleotides constructs to be synthesized. In oneembodiment, oligonucleotides are short nucleic acid molecules. Forexample, oligonucleotides may be from 10 to about 300 nucleotides, from20 to about 400 nucleotides, from 30 to about 500 nucleotides, from 40to about 600 nucleotides, or more than about 600 nucleotides long.However, shorter or longer oligonucleotides may be used.Oligonucleotides may be designed to have different length. In someembodiments, the sequence of the polynucleotide construct may be dividedup into a plurality of shorter sequences that can be synthesized inparallel and assembled into a single or a plurality of desiredpolynucleotide constructs using the methods described herein.

In some embodiments, the assembly procedure may include several paralleland/or sequential reaction steps in which a plurality of differentnucleic acids or oligonucleotides are synthesized or immobilized,amplified, and are combined in order to be assembled (e.g., by extensionor ligation as described herein) to generate a longer nucleic acidproduct to be used for further assembly, cloning, or other applications(see U.S. provisional application 61/235,677 and PCT applicationPCT/US09/55267, now International Publication No. WO2010/025310, whichare incorporated herein by reference in their entirety). Amplificationand assembly strategies provided herein can be used to generate verylarge libraries representative of many different nucleic acid sequencesof interest.

In some embodiments, methods of assembling libraries containing nucleicacids having predetermined sequence variations are provided herein.Assembly strategies provided herein can be used to generate very largelibraries representative of many different nucleic acid sequences ofinterest. In some embodiments, libraries of nucleic acid are librariesof sequence variants. Sequence variants may be variants of a singlenaturally-occurring protein encoding sequence. However, in someembodiments, sequence variants may be variants of a plurality ofdifferent protein-encoding sequences.

Accordingly, one aspect of the technology provided herein relates to thedesign of assembly strategies for preparing precise high-density nucleicacid libraries. Another aspect of the technology provided herein relatesto assembling precise high-density nucleic acid libraries. Aspects ofthe technology provided herein also provide precise high-density nucleicacid libraries. A high-density nucleic acid library may include morethat 100 different sequence variants (e.g., about 10² to 10³; about 10³to 10⁴; about 10⁴ to 10⁵; about 10⁵ to 10⁶; about 10⁶ to 10⁷; about 10⁷to 10⁸; about 10⁸ to 10⁹; about 10⁹ to 10¹⁰; about 10¹⁰ to 10¹¹; about10¹¹ to 10¹²; about 10¹² to 10¹³; about 10¹³ to 10¹⁴; about 10¹⁴ to10¹⁵; or more different sequences) wherein a high percentage of thedifferent sequences are specified sequences as opposed to randomsequences (e.g., more than about 50%, more than about 60%, more thanabout 70%, more than about 75%, more than about 80%, more than about85%, more than about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or more of thesequences are predetermined sequences of interest).

Oligonucleotides can be used as building blocks for DNA synthesis andcan be synthesized in single strands on a solid support (e.g. microarraychip surface) using inkjet and other technologies. In nucleic acidassembly process, synthetic oligonucleotides can be used to assemble oramplify DNA into large size DNA constructs. During enzymaticamplification, the error in sequence is faithfully replicated. As aresult, DNA population synthesized by this method contains botherror-free and error-prone sequences. In some embodiments, sincesynthetic oligonucleotides can contain incorrect sequences due to errorsintroduced during oligonucleotide synthesis, it can be useful to removenucleic acid fragments that have incorporated one or moreerror-containing oligonucleotides during assembly. In some embodiments,one or more assembled nucleic acid fragments may be sequenced todetermine whether they contain the predetermined sequence or not. Thisprocedure allows fragments with the correct sequence to be identified.In other embodiments, other techniques may be used to remove errorcontaining nucleic acid fragments. Such nucleic acid fragments can benascently synthesized oligonucleotides or assembled nucleic acidpolymers. It should be appreciated that error containing-nucleic acidscan be double-stranded homoduplexes having the error on both strands(i.e., incorrect complementary nucleotide(s), deletion(s), oraddition(s) on both strands), because the assembly procedure may involveone or more rounds of polymerase extension (e.g., during assembly orafter assembly to amplify the assembled product) during which an inputnucleic acid containing an error may serve as a template therebyproducing a complementary strand with the complementary error. Incertain embodiments, a preparation of double-stranded nucleic acidfragments may be suspected to contain a mixture of nucleic acids thathave the correct sequence and nucleic acids that incorporated one ormore sequence errors during assembly. In some embodiments, sequenceerrors may be removed using a technique that involves denaturing andreannealing the double-stranded nucleic acids. In some embodiments,single strands of nucleic acids that contain complementary errors may beunlikely to reanneal together if nucleic acids containing eachindividual error are present in the nucleic acid preparation at a lowerfrequency than nucleic acids having the correct sequence at the sameposition. Rather, error containing single strands can reanneal with acomplementary strand that does not contain any error or that containsone or more different errors (e.g. errors at different positions). As aresult, error-containing strands can end up in the form of heteroduplexmolecules in the reannealed reaction product. Nucleic acid strands thatare error-free may reanneal with error-containing strands or with othererror-free strands. Reannealed error-free strands form homoduplexes inthe reannealed sample. Accordingly, by removing heteroduplex moleculesfrom the reannealed preparation of nucleic acid fragments, the amount orfrequency of error containing nucleic acids can be reduced.

Heteroduplex formation thus takes place through a process that can beunderstood as shuffling, by which nucleic acid strands from differentpopulations can be hybridized with one another so that perfect match andmismatch-containing duplexes can be formed. Suitable method for removingheteroduplex molecules include chromatography, electrophoresis,selective binding of heteroduplex molecules, etc. One requirement forthe selective binding agent for use in this process is that it bindspreferentially to double stranded DNA having a sequence mismatch betweenthe two strands. In some embodiments, such agent can be a mismatchendonuclease. The term “endonuclease” in general refers to an enzymethat can cleave DNA internally. The term “mismatch” or “base pairmismatch” indicates a base pair combination that generally does not formin nucleic acids according to Watson and Crick base pairing rules. Forexample, when dealing with the bases commonly found in DNA, namelyadenine, guanine, cytosine and thymidine, base pair mismatches are thosebase combinations other than the A-T and G-C pairs normally found inDNA. As described herein, a mismatch may be indicated, for example asC/C meaning that a cytosine residue is found opposite another cytosine,as opposed to the proper pairing partner, guanine. As used herein“heteroduplex” oligonucleotide refers to a double-strandedoligonucleotide formed by annealing single strands oligonucleotideswhere the strands, when annealed have unpaired regions such as base-pairmismatch, insertion/deletion loop(s) and or nucleotide(s) gap(s). In oneaspect, the invention relates to a method for producing high fidelityoligonucleotides on a solid support. The method includes synthesizing afirst plurality of oligonucleotides in a chain extension reaction usinga second plurality of oligonucleotides as templates. The secondplurality of oligonucleotides are immobilized on a solid support andlikely comprises both error-free and error-containing oligonucleotideshaving a sequence error at an error-containing position. The chainextension reaction produces a first plurality of double-strandedoligonucleotides or duplexes (homoduplexes and/or heteroduplexes). Themethod also includes melting off or denaturing the first plurality ofduplexes to release the first plurality of oligonucleotides from thesecond plurality of oligonucleotides, wherein the first plurality ofoligonucleotides comprise error-free oligonucleotides that are free oferror at the error-containing position of the error-containingoligonucleotide. The method further includes contacting the firstplurality of oligonucleotides with the second plurality ofoligonucleotides under hybridization or annealing conditions to form asecond plurality of duplexes. The second plurality of duplexes comprisea mismatch-containing heteroduplex formed between the error-containingoligonucleotide and one of the error-free oligonucleotides. The methodalso includes removing at least a portion of the mismatch-containingheteroduplex by a mismatch recognizing and cleaving component, therebyproducing high fidelity oligonucleotides. In some embodiments, themethod further includes after the removing step, selectively meltingaway truncations.

FIG. 1 shows an exemplary method for producing high fidelityoligonucleotides on a DNA microarray where heteroduplex formation isfollowed by mismatch cleavage. The microarray contains chemicallysynthesized oligonucleotides that are immobilized on chip surface 10.The chemically synthesized oligonucleotides, as discussed above, likelycontain both error-free template strand 11 and error-prone templatestrand 12. By way of example, in a chain extension reaction (e.g., PCR)using primer 13 (e.g., a universal amplification primer), the chemicallysynthesized oligonucleotides can serve as template strands for producingcomplementary strands. The resulting products can include error-freecomplementary strand 14 (complementary to error-free template strand 11)and error-prone amplified complementary strand 15 (complementary toerror-prone template strand 12). Under melting conditions (e.g., anincreased temperature at solid support or chip surface 10), thecomplementary strands are separated from the template strands. Aftershuffling, heteroduplex 16 can be formed between an error-prone templatestrand and an error-free complementary strand. Heteroduplex 16 can thenbe recognized and cleaved by a component 17 (e.g., Surveyorendonuclease). Subsequent removal of cleaved, error-prone duplexes canresult in an error-free chip surface 18.

Heteroduplex recognition and cleavage can be achieved by applying amismatch binding agent to the reaction mix. In some embodiments, themismatch binding agent is a mismatch specific endonuclease. In someembodiment, a mismatch-binding protein tethered or fused to a nucleasecan be used.

One preferred mismatch endonuclease is a CEL1 endonuclease which has ahigh specificity for insertions, deletions and base substitutionmismatches and can detect two polymorphisms which are five nucleotidesapart form each other. CEL1 is a plant-specific extracellularglycoprotein that can cleave heteroduplex DNA at all possible singlenucleotide mismatches, 3′ to the mismatches (Oleykowski Calif. et al,1998, Nucleic Acids Res. 26: 4596-4602). CEL1 is useful in mismatchdetection assays that rely on nicking and cleaving duplex DNA atinsertion/deletion and base substitution mismatches. In an exemplaryembodiment, a Surveyor™ Nuclease (Transgenomic Inc.) may be added to areaction volume containing the oligonucleotide duplexes. Surveyor™Nuclease is a mismatch specific endonuclease that cleaves all types ofmismatches such as single nucleotide polymorphisms, small insertions ordeletions. Addition of the endonuclease results in the cleavage of thedouble-stranded oligonucleotides at the site or region of the mismatch.The remaining portion of the oligonucleotide duplexes can then be meltedat a lower and less stringent temperature (e.g. stringent melt) neededto distinguish a single base mismatch. In some embodiments, theerror-free oligonucleotides are released in solution.

The mismatch-containing duplexes from the population can be preferablycleaved by mismatch specific CEL1 endonuclease. In some embodiments,other mismatch binding proteins that selectively (e.g., specifically)bind to heteroduplex nucleic acid molecules may be used. One exampleincludes using MutS, a MutS homolog, or a combination thereof to bind toheteroduplex molecules. MutS from Thermus aquaticus can be purchasecommercially from the Epicenter Corporation, Madison, Wis., Catalog No.SP72100 and SP72250. The gene sequence for the protein is also known andpublished in Biswas and Hsieh, Jour. Biol. Chem. 271:5040-5048 (1996)and is available in GenBank, accession number U33117. In E. coli, theMutS protein, which appears to function as a homodimer, serves as amismatch recognition factor. In eukaryotes, at least three MutS Homolog(MSH) proteins have been identified; namely, MSH2, MSH3, and MSH6, andthey form heterodimers. For example in the yeast, Saccharomycescerevisiae, the MSH2-MSH6 complex (also known as MutSα) recognizes basemismatches and single nucleotide insertion/deletion loops, while theMSH2-MSH3 complex (also known as MutSβ) recognizes insertions/deletionsof up to 12-16 nucleotides, although they exert substantially redundantfunctions. A mismatch binding protein may be obtained from recombinantor natural sources. A mismatch binding protein may be heat-stable. Insome embodiments, a thermostable mismatch binding protein from athermophilic organism may be used. Examples of thermostable DNA mismatchbinding proteins include, but are not limited to: Tth MutS (from Thermusthermophilus); Taq MutS (from Thermus aquaticus); Apy MutS (from Aquifexpyrophilus); Tma MutS (from Thermotoga maritima); any other suitableMutS; or any combination of two or more thereof.

In some embodiments, small molecules capable to bind to specificnucleotide mismatches can be designed and synthesized. For example,dimeric napthyridine 1, a synthetic ligand that binds to a G-G mismatch.A cocktail of such ligands which, in combination, recognizes allpossible mismatches could replace CEL1. Other protein agents that candifferentiate between matched and unmatched duplexes could also be used.For example, the T7 endonuclease I will specifically cleave a DNA strandat a mismatch, and it would be possible to use this enzyme as acatalytic destroyer of mismatched sequences or to inactivate thecleavage function of this enzyme for use in this process as a mismatchbinding agent. T4 endonuclease VII will specifically bind and cleave DNAat duplex mismatches and a mutant version of this enzyme has alreadybeen engineered that lacks the nuclease activity but retains the abilityto bind mutant duplex DNA molecules (Golz and Kemper, Nucleic AcidsResearch, 27:e7 (1999)). SP nuclease is a highly active nuclease fromspinach that incises all mismatches except those containing a guanineresidue, and this enzyme could also be engineered to remove the cleavageactivity or used directly. Two or more of these binding agents could becombined to either provide further stringency to the filtration or tocover all types of sequence errors if one agent does not bind to allpossible mismatches.

Some embodiments of the device and methods provided herein useoligonucleotides that are immobilized on a surface or substrate. As usedherein the term “support” and “substrate” are used interchangeably andrefers to a porous or non-porous solvent insoluble material on whichpolymers such as nucleic acids are synthesized or immobilized. As usedherein “porous” means that the material contains pores havingsubstantially uniform diameters (for example in the nm range). Porousmaterials include paper, synthetic filters etc. In such porousmaterials, the reaction may take place within the pores. The support canhave any one of a number of shapes, such as pin, strip, plate, disk,rod, bends, cylindrical structure, particle, including bead,nanoparticles and the like. The support can have variable widths. Thesupport can be hydrophilic or capable of being rendered hydrophilic andincludes inorganic powders such as silica, magnesium sulfate, andalumina; natural polymeric materials, particularly cellulosic materialsand materials derived from cellulose, such as fiber containing papers,e.g., filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly (4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), polyvinylidene difluoride (PVDF) membrane, glass,controlled pore glass, magnetic controlled pore glass, ceramics, metals,and the like etc.; either used by themselves or in conjunction withother materials. In some embodiments, oligonucleotides are synthesizedon an array format. For example, single-stranded oligonucleotides aresynthesized in situ on a common support wherein each oligonucleotide issynthesized on a separate or discrete feature (or spot) on thesubstrate. In preferred embodiments, single stranded oligonucleotidesare bound to the surface of the support or feature. As used herein theterm “array” refers to an arrangement of discrete features for storing,routing, amplifying and releasing oligonucleotides or complementaryoligonucleotides for further reactions. In a preferred embodiment, thesupport or array is addressable: the support includes two or morediscrete addressable features at a particular predetermined location (i.e., an “address”) on the support. Therefore, each oligonucleotidemolecule of the array is localized to a known and defined location onthe support. The sequence of each oligonucleotide can be determined fromits position on the support. Moreover, addressable supports or arraysenable the direct control of individual isolated volumes such asdroplets. The size of the defined feature is chosen to allow formationof a microvolume droplet on the feature, each droplet being keptseparate from each other. As described herein, features are typically,but need not be, separated by interfeature spaces to ensure thatdroplets between two adjacent features do not merge. Interfeatures willtypically not carry any oligonucleotide on their surface and willcorrespond to inert space. In some embodiments, features andinterfeatures may differ in their hydrophilicity or hydrophobicityproperties. In some embodiments, features and interfeatures may comprisea modifier as described herein.

Arrays may be constructed, custom ordered or purchased from a commercialvendor (e.g., Agilent, Affymetrix, Nimblegen). Oligonucleotides areattached, spotted, immobilized, surface-bound, supported or synthesizedon the discrete features of the surface or array as described above.Oligonucleotides may be covalently attached to the surface or depositedon the surface. Various methods of construction are well known in theart e.g. maskless array synthesizers, light directed methods utilizingmasks, flow channel methods, spotting methods etc.

In some embodiments, construction and/or selection oligonucleotides maybe synthesized on a solid support using maskless array synthesizer(MAS). Maskless array synthesizers are described, for example, in PCTapplication No. WO 99/42813 and in corresponding U.S. Pat. No.6,375,903. Other examples are known of maskless instruments which canfabricate a custom DNA microarray in which each of the features in thearray has a single-stranded DNA molecule of desired sequence.

Other methods for synthesizing construction and/or selectionoligonucleotides include, for example, light-directed methods utilizingmasks, flow channel methods, spotting methods, pin-based methods, andmethods utilizing multiple supports. Light directed methods utilizingmasks (e.g., VLSIPS™ methods) for the synthesis of oligonucleotides isdescribed, for example, in U.S. Pat. Nos. 5,143,854, 5,510,270 and5,527,681. These methods involve activating predefined regions of asolid support and then contacting the support with a preselected monomersolution. Selected regions can be activated by irradiation with a lightsource through a mask much in the manner of photolithography techniquesused in integrated circuit fabrication. Other regions of the supportremain inactive because illumination is blocked by the mask and theyremain chemically protected. Thus, a light pattern defines which regionsof the support react with a given monomer. By repeatedly activatingdifferent sets of predefined regions and contacting different monomersolutions with the support, a diverse array of polymers is produced onthe support. Other steps, such as washing unreacted monomer solutionfrom the support, can be optionally used. Other applicable methodsinclude mechanical techniques such as those described in U.S. Pat. No.5,384,261.

Additional methods applicable to synthesis of construction and/orselection oligonucleotides on a single support are described, forexample, in U.S. Pat. No. 5,384,261. For example, reagents may bedelivered to the support by either (1) flowing within a channel definedon predefined regions or (2) “spotting” on predefined regions. Otherapproaches, as well as combinations of spotting and flowing, may beemployed as well. In each instance, certain activated regions of thesupport are mechanically separated from other regions when the monomersolutions are delivered to the various reaction sites. Flow channelmethods involve, for example, microfluidic systems to control synthesisof oligonucleotides on a solid support. For example, diverse polymersequences may be synthesized at selected regions of a solid support byforming flow channels on a surface of the support through whichappropriate reagents flow or in which appropriate reagents are placed.Spotting methods for preparation of oligonucleotides on a solid supportinvolve delivering reactants in relatively small quantities by directlydepositing them in selected regions. In some steps, the entire supportsurface can be sprayed or otherwise coated with a solution, if it ismore efficient to do so. Precisely measured aliquots of monomersolutions may be deposited dropwise by a dispenser that moves fromregion to region.

Pin-based methods for synthesis of oligonucleotides on a solid supportare described, for example, in U.S. Pat. No. 5,288,514. Pin-basedmethods utilize a support having a plurality of pins or otherextensions. The pins are each inserted simultaneously into individualreagent containers in a tray. An array of 96 pins is commonly utilizedwith a 96-container tray, such as a 96-well microtiter dish. Each trayis filled with a particular reagent for coupling in a particularchemical reaction on an individual pin. Accordingly, the trays willoften contain different reagents. Since the chemical reactions have beenoptimized such that each of the reactions can be performed under arelatively similar set of reaction conditions, it becomes possible toconduct multiple chemical coupling steps simultaneously.

In another embodiment, a plurality of oligonucleotides may besynthesized on multiple supports. One example is a bead based synthesismethod which is described, for example, in U.S. Pat. Nos. 5,770,358;5,639,603; and 5,541,061. For the synthesis of molecules such asoligonucleotides on beads, a large plurality of beads is suspended in asuitable carrier (such as water) in a container. The beads are providedwith optional spacer molecules having an active site to which iscomplexed, optionally, a protecting group. At each step of thesynthesis, the beads are divided for coupling into a plurality ofcontainers. After the nascent oligonucleotide chains are deprotected, adifferent monomer solution is added to each container, so that on allbeads in a given container, the same nucleotide addition reactionoccurs. The beads are then washed of excess reagents, pooled in a singlecontainer, mixed and re-distributed into another plurality of containersin preparation for the next round of synthesis. It should be noted thatby virtue of the large number of beads utilized at the outset, therewill similarly be a large number of beads randomly dispersed in thecontainer, each having a unique oligonucleotide sequence synthesized ona surface thereof after numerous rounds of randomized addition of bases.An individual bead may be tagged with a sequence which is unique to thedouble-stranded oligonucleotide thereon, to allow for identificationduring use.

In yet another embodiment, a plurality of oligonucleotides may beattached or synthesized on nanoparticles. Nanoparticles includes but arenot limited to metal (e.g., gold, silver, copper and platinum),semiconductor (e.g., CdSe, CdS, and CdS coated with ZnS) and magnetic(e.g., ferromagnetite) colloidal materials. Methods to attacholigonucleotides to the nanoparticles are known in the art. In anotherembodiment, nanoparticles are attached to the substrate. Nanoparticleswith or without immobilized oligonucleotides can be attached tosubstrates as described in, e.g., Grabar et al., Analyt. Chem., 67,73-743 (1995); Bethell et al., J. Electroanal. Chem., 409, 137 (1996);Bar et al., Langmuir, 12, 1172 (1996); Colvin et al., J. Am. Chem. Soc.,114, 5221 (1992). Naked nanoparticles may be first attached to thesubstrate and oligonucleotides can be attached to the immobilizednanoparticles.

Pre-synthesized oligonucleotide and/or polynucleotide sequences may beattached to a support or synthesized in situ using light-directedmethods, flow channel and spotting methods, inkjet methods, pin-basedmethods and bead-based methods set forth in the following references:McGall et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:13555; SyntheticDNA Arrays In Genetic Engineering, Vol. 20:111, Plenum Press (1998);Duggan et al. (1999) Nat. Genet. S21:10; Microarrays: Making Them andUsing Them In Microarray Bioinformatics, Cambridge University Press,2003; U.S. Patent Application Publication Nos. 2003/0068633 and2002/0081582; U.S. Pat. Nos. 6,833,450, 6,830,890, 6,824,866, 6,800,439,6,375,903 and 5,700,637; and PCT Publication Nos. WO 04/031399, WO04/031351, WO 04/029586, WO 03/100012, WO 03/066212, WO 03/065038, WO03/064699, WO 03/064027, WO 03/064026, WO 03/046223, WO 03/040410 and WO02/24597; the disclosures of which are incorporated herein by referencein their entirety for all purposes. In some embodiments, pre-synthesizedoligonucleotides are attached to a support or are synthesized using aspotting methodology wherein monomers solutions are deposited dropwiseby a dispenser that moves from region to region (e.g. ink jet). In someembodiments, oligonucleotides are spotted on a support using, forexample, a mechanical wave actuated dispenser.

Methods and devices provided herein involve amplification and/or smallassembly reaction volumes such as microvolumes, nanovolumes, picovolumesor sub-picovolumes. Accordingly, aspects of the invention relate tomethods and devices for amplification and/or assembly of polynucleotidesin small volume droplets on separate and addressable features of asupport. For example, a plurality of oligonucleotides complementary tosurface-bound single stranded oligonucleotides is synthesized in apredefined reaction microvolume of solution by template-dependantsynthesis. In some embodiments, predefined reaction microvolumes ofbetween about 0.5 pL and about 100 nL may be used. However, smaller orlarger volumes may be used. In some embodiments, a mechanical waveactuated dispenser may be used for transferring volumes of less than 100nL, less than 10 nL, less than 5 nL, less than 100 pL, less than 10 pL,or about 0.5 pL or less. In some embodiments, the mechanical waveactuated dispenser can be a piezoelectric inkjet device or an acousticliquid handler. In a preferred embodiment, a piezoelectric inkjet deviceis used and can deliver picoliter solutions in a very precise manner ona support as described herein.

Aspects of the invention relate to the manipulation of sub-microvolumeson a substrate and to the control of the movement of micro-volumes on asubstrate. It is a well-known phenomenon that the surfaces of mostnormally solid substrates, when contacted with a solution, have acharacteristic degree of non-wettability. That is, aqueous solutions donot spread on the solid surface but contract to form droplets.Accordingly, preferable supports have surface properties, primarilysurface tension and wettability properties that allow droplet formationwhen small volumes are dispensed onto the addressable feature. In someembodiments, the microvolume is bounded completely or almost completelyby free surface forming a droplet or microdrop. One skilled in the artwill understand that the shape of the droplet will be governed andmaintained by the contact angle of the liquid/solid interaction, surfacetension of the liquid as well as by surface energy. Adhesive forcesbetween a liquid and solid will cause a liquid drop to spread across thesurface whereas cohesive forces within the liquid will cause the drop toball up and avoid contact with the surface. For liquid, the surfaceenergy density is identical to the surface tension. Surface tension isthat property of matter, due to molecular forces, which exists in thesurface film of all liquids and tends to bring the contained volume intoa form having the least possible superficial area. In some embodiments,the surface is partitioned into discrete regions where the surfacecontact angles of the discrete region differ for the fluid of interest.As used herein the term “contact angle” refers to a quantitative measureof the wetting of a solid by a liquid. A contact angle is defined as theangle formed by a liquid at the three phase boundary where vapor (gas,e.g., atmosphere), liquid and solid intersect. For example, in the caseof a micro-volume droplet dispensed on a horizontal flat surface, theshape of the micro-volume droplet will be determined by the Youngequilibrium equation:

0=γ_(SV)−γ_(SL)−γ cos θ_(C)

wherein γ_(SV) is the solid-vapor interfacial energy; γ_(SL) is thesolid-liquid interfacial energy and γ is the liquid-vapor energy (i.e.surface tension) and θ_(C) is the equilibrium contact angle.

It will be understood that for contact angle values θ_(C) less than 90°,the liquid will spread onto the solid surface. For example, veryhydrophilic surfaces have a contact angle of 0° to about 30°. In thecase of aqueous solutions and highly hydrophilic support, the contactangle θ_(C) will be close to 0°, and the aqueous solution or dropletwill completely spread out on the solid surface (i.e., complete wettingof the surface). If complete wetting does not occur, the liquid willfrom a droplet. On the contrary, for contact angle values θ_(C) equal toor greater than 90°, the liquid will rest on the surface and form adroplet on the solid surface. The shape of the droplet is determined bythe value of the contact angle. In the case of aqueous solutions andhighly hydrophobic surfaces, liquid will bead up. In some embodiments,the support is chosen to have a surface energy and surface contact anglethat do not allow the droplets to spread beyond the perimeter of thefeature. Furthermore, on an ideal surface the droplets will return totheir original shapes if they are disturbed, for example after additionof a miscible or non-miscible solution. In some embodiments, the surfaceis partitioned into regions where the surface contact angles of theregions differ for the liquid of interest. In some embodiments, thesesregions correspond to the discrete features of the substrate. In apreferred embodiment, the surface is partitioned into regions bymodifiers. Modifiers may be added to specific locations of thesubstrate's surface. In some cases, the surface will be partitioned intoregions comprising modifiers and non-modifier surface areas. In someembodiments, the non-modifier regions correspond to the unmodifiedsubstrate. Yet, in other embodiments, the non-modifiers regionscorrespond to a surface of any arbitrary modification or any modifierthat is different than the modifier at a region that corresponds to afeature of a support. In some embodiments, the modifiers are oligomers.For example, the modifiers correspond to nucleic acids and are modifyinga set of discrete features of the substrate. Modifiers can havecircular, square, trapezoid, or any geometrical shape or any combinationthereof. In some embodiments, modifiers are arranged in a grid-likepattern or in any other different configurations. The pattern is notrestricted to any design. For example, the modifiers may be arranged ina randomly formed pattern. Patterning may be formed by any process knownin the art. For example, arranged patterning or random patterning may beformed by processes such as block co-polymer surface self assembly. Inother embodiments, the substrate surface is partitioned into regions byat least two different modifiers regions as discussed herein. In someembodiments, the surface contact angle of the modifiers (θ_(M)) isdifferent than the surface contact angle of the non-modifier region(θ_(NM)). For example, the surface contact angle of the modifiers may begreater than the surface contact angle of the surface or thenon-modifier regions (θ_(M)>θ_(NM)). Alternatively, the surface contactangle of the non-modifier regions is greater than the surface contactangle of the modifiers (θ_(M)<θ_(NM)). In the context of aqueoussolutions, the modifiers surfaces may be more hydrophilic than thesurface of the non-modifiers regions (i.e. surface contact angle of themodifiers is smaller than the surface contact angle of the surface ornon-modifier regions). Alternatively, the modifiers surfaces may be morehydrophobic than the surface of the non-modifiers surface regions (i.e.,surface contact angle of the modifiers is smaller than the surfacecontact angle of the surface or non-modifier regions). In an exemplaryembodiment, modifiers are oligonucleotides and the surface of themodifier regions is more hydrophilic than the surface of thenon-modifier regions. In other embodiments, the totality or asubstantial part of the support or surface is covered with at least twodifferent modifiers. The at least two different modifiers may bepatterned as described above. For example, the different modifiers cancover the surface in an alternative pattern. On should appreciate thatthe support surface may be covered with a plurality of modifiers thatare disposed on the surface to form a hydrophilic gradient. In someembodiments, each modifier has a different contact angle than theadjacent modifier. In some embodiments, the surface is partitioned witha plurality of different modifiers, the plurality of first modifiersbeing more hydrophilic than the at least one second modifier, theplurality of first modifiers having each a slightly different contactangle than the next first modifier. For example, the contact angle ofeach of the plurality of first modifier may differ by at least about 1°,2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10° 11°, 12°, 13°, 14°, 15°, 16°, 17°,18°, 19°, 20°, 25°, 30° or more from that of the next first modifier.The plurality of first modifiers therefore forms a hydrophilic gradientand a predetermined path along which a droplet can be moved bysurface-tension manipulation.

According to some aspects of the invention, the difference in surfacecontact angles between two different modifiers or a modifier and thenon-modifier surface creates a virtual “wall”. As used herein the termsmaller contact angle (SCA) refers to the surface or modifier havingsmaller contact angle and the term higher contact angle (HCA) refers tothe surface or modifier having higher contact angle. In the context ofaqueous solutions, SCA are more hydrophilic than HCA. In someembodiments, HCA values are at least 20°, at least 30°, at least 35°higher than SCA. Accordingly, liquid volumes can be formed and isolatedon such surfaces. For example, if the surface contact angle of themodifier is greater than the non-modifier surface contact angle, liquidvolumes will form a droplet between two modifiers regions. One wouldappreciate that depending on liquid volume deposited onto the surfaceand the difference of contact values between modifiers, the droplet canoccupy a single region of small contact angle (SCA) or multiple regions.For example, the liquid volume may occupy 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more SCA regions. Accordingly, the liquid can occupy a footprintcorresponding to one or more SCA. The footprint may then encompass oneor more HCA. In some embodiments, to ensure that two droplets or smallisolated volumes will not merge, liquid volumes are placed sufficientlyapart from each others. For example, the spacing between two isolatedvolumes may comprise at least one, at least two, at least three, atleast four, at least five, at least six, at least seven, at least eight,at least nine, at least ten HCA regions or modifiers regions. Placingthe liquid volumes sufficiently apart also allows for keeping liquidvolumes isolated during fluctuation of temperature such as duringthermocycling. Because surface tension usually decreases with theincrease of temperature, droplets may spread or move on the surface whenthe temperature of the support or of the liquid volume is raised. Itwill be appreciate that if the liquid volumes are kept sufficientlyapart, liquid volumes will remain isolated and will not merge withadjacent liquid volumes during fluctuation of the temperature.

In one aspect of the invention, methods and devices are provided forprocessing independently one or more plurality of oligonucleotides in atemperature dependent manner at addressable features in isolated liquidvolumes. In some embodiments, the method is conducted in a manner toprovide a set of predefined single-stranded oligonucleotides orcomplementary oligonucleotides sequences for further specified reactionsor processing. One should appreciate that each features beingindependently addressable, each reaction can be processed independentlywithin a predefined isolated liquid volume or droplet on a discretefeature (e.g. virtual chamber). In some embodiments, the arrays arestored dry for subsequent reactions. In a preferred embodiment, supportimmobilized oligonucleotides can be hydrated independently with anaqueous solution. Aqueous solutions include but is not limited to water,buffer, primers, master mix, release chemicals, enzymes, or anycombination thereof. Aqueous solution can be spotted or jetted ontospecific surface location(s) corresponding to the discrete feature(s).Subsequently, miscible as well as non-miscible solution or aqueous gelcan be deposited in the same fashion. Alternatively, a mechanical waveactuated dispenser can be used for transferring small volumes of fluids(e.g., picoliter or sub-picoliter). A mechanical wave actuated dispensercan be a piezoelectric inkjet device or an acoustic liquid handler. Apiezoelectric inkjet device can eject fluids by actuating apiezoelectric actuation mechanism, which forces fluid droplets to beejected. Piezoelectrics in general have good operating bandwidth and cangenerate large forces in a compact size. Some of the commerciallyavailable piezoelectric inkjet microarraying instruments include thosefrom Perkin Elmer (Wellesley, Mass.), GeSim (Germany) and MicroFab(Plano, Tex.). Typical piezoelectric dispensers can create droplets inthe picoliter range and with coefficient of variations of 3-7%.Inkjetting technologies and devices for ejecting a plurality of fluiddroplets toward discrete features on a substrate surface for depositionthereon have been described in a number of patents such as U.S. Pat.Nos. 6,511,849; 6,514,704; 6,042,211; 5,658,802, the disclosure of eachof which is incorporated herein by reference.

In one embodiment, the fluid or solution deposition is performed usingan acoustic liquid handler or ejector. Acoustic devices are non-contactdispensing devices able to dispense small volume of fluid (e.g.picoliter to microliter), see for example Echo 550 from Labcyte (CA),HTS-01 from EDC Biosystems. Acoustic technologies and devices foracoustically ejecting a plurality of fluid droplets toward discretesites on a substrate surface for deposition thereon have been describedin a number of patents such as U.S. Pat. Nos. 6,416,164; 6,596,239;6,802,593; 6,932,097; 7,090,333 and US Patent Application 2002-0037579,the disclosure of each of which is incorporated herein by reference. Theacoustic device includes an acoustic radiation generator or transducerthat may be used to eject fluid droplets from a reservoir (e.g.microplate wells) through a coupling medium. The pressure of the focusedacoustic waves at the fluid surface creates an upwelling, therebycausing the liquid to urge upwards so as to eject a droplet, for examplefrom a well of a source plate, to a receiving plate positioned above thefluid reservoir. The volume of the droplet ejected can be determined byselecting the appropriate sound wave frequency.

In some embodiments, the source plate comprising water, buffer, primers,master mix, release chemicals, enzymes, or any combination thereof andthe destination plates comprising the oligonucleotides orpolynucleotides are matched up to allow proper delivery or spotting ofthe reagent to the proper site. The mechanical wave actuated dispensermay be coupled with a microscope and/or a camera to provide positionalselection of deposited spots. A camera may be placed on both sides ofthe destination plate or substrate. A camera may be used to register tothe positioning on the array especially if the nucleic acid is coupledwith a fluorescent label.

One should appreciate that when manipulating small liquid volumes suchas picoliters and nanoliters, the smaller the droplet, the faster itwill evaporate. Therefore, aspects of the invention relate to methodsand devices to limit, retard or prevent water or solvent evaporation. Insome embodiments, the discrete features or a subset of discrete featurescan be coated with a substance capable of trapping or capturing water.In other embodiments, the water-trapping material can be spin-coatedonto the support. Different materials or substances can be used to trapwater at specific locations. For example, the water trapping substancemay be an aqueous matrix, a gel, a colloid or any suitable polymer. Insome embodiments, the material is chosen to have a melting point thatallows it to remain solid or semi-solid (e.g. gel) at the reactiontemperatures such as denaturing temperatures or thermocyclingtemperatures. Water trapping materials include but are not limited tocolloidal silica, peptide gel, agarose, solgel and polydimethylsiloxane.In an exemplary embodiment, Snowtex® colloidal silica (Nissan Chemical)may be used. Snowtex colloidal silica is composed of mono-dispersed,negatively charged, amorphous silica particles in water. Snowtexcolloidal silica can be applied as dry gel or as an hydrated gel ontothe surface. In a preferred embodiment, the water trapping substance isspotted at discrete features comprising surface-bound oligonucleotides.Alternatively, oligonucleotides can be synthesized on the particles ornanoparticles (e.g. colloidal particles, Snowtex colloidal silica) andthe particles or nanoparticles can be dispensed to the discrete featuresof the surface. In some embodiments, the water trapping substance isspotted on the discrete features of the support using a mechanicaldevice, an inkjet device or an acoustic liquid handler.

One should appreciate, that evaporation can also be limited by forming aphysical barrier between the surface of the droplet and the atmosphere.For example, a non-miscible solution can be overlaid to protect thedroplet from evaporation. In some embodiments, a small volume of thenon-miscible solution is dispensed directly and selectively at discretelocation of the substrate such as features comprising a droplet. In someother embodiments, the non-miscible solution is dispensed onto a subsetof features comprising a droplet. In other embodiments, the non-misciblesolution is applied uniformly over the surface of the array forming anon-miscible bilayer in which the droplets are trapped. The non-misciblebilayer can then be evaporated to form a thin film over the surface orover a substantial part of the surface of the droplet. The non-misciblesolution includes, but is not limited to, mineral oil, vegetable oil,silicone oil, paraffin oil, natural or synthetic wax, organic solventthat is immiscible in water or any combination thereof. One skilled inthe art will appreciate that depending on the composition of the oils,some oils may partially or totally solidify at or below roomtemperature. In some embodiments, the non-miscible solution may be anatural or synthetic wax such as paraffin hydrocarbon. Paraffin is analkane hydrocarbon with the general formula C—H_(2n+2). Depending on thelength of the molecule, paraffin may appear as a gas, a liquid or asolid at room temperature. Paraffin wax refers to the solids with20≦n≦40 and has a typical melting point between about 47° C. to 64° C.Accordingly, in some embodiments, the support may be stored capped witha wax. Prior to use, heat may be applied to the support to allow the waxto turn into a liquid wax phase coating the support.

In some aspects of the invention, in subsequent steps, aqueous solutionmay be added to the droplet having a non-miscible solution at itssurface. Solvent or aqueous solution may be added, for example, toinitiate a reaction, to adjust a volume, to adjust a pH, to increase ordecrease a solute concentration, etc. . . . One would appreciate thatthe solvent or aqueous solution can penetrate the non-miscible layerusing different mechanisms. For example, if using an inkjet head device,the aqueous solution is ejected and the physical momentum of the ejecteddroplet will enable the aqueous solution to cross the non-misciblelayer. Other mechanisms may employ additional forces, such as forexample magnetic and/or electrostatic forces and/or optical forces. Theoptical and magnetic forces can be created simultaneously orindependently of one another. Furthermore, the mechanism can utilizecoupled magneto-optical tweezers. In some embodiments, the aqueoussolution to be dispensed contains magnetic nanoparticles and a magneticforce can be used to help penetration of the non-miscible layer.Alternatively, the aqueous solution carries an electrostatic charge andan external applied electric field can be used to achieve penetration ofthe non-miscible layer.

Yet, in another aspect of the invention, the size of the droplet iscontinuously or frequently monitored. One should appreciate that thesize of the droplet is determined by the volume and by the surfacetension of the solution. Accordingly, loss of volume can be detected bya decrease of the droplet footprint or radius of the droplet footprint.For example, using an optical monitoring system, through a microscopelens and camera system, the size or footprint of the droplet can bedetermined and the volume of the droplet can be calculated. In someembodiments, the volume of the droplet or the radius of the droplet ismonitored every second or every millisecond. One would appreciate thatthe magnitude of the evaporation rate of the water from the droplet ofinterest depends in part on the temperature and thus increases withincreasing temperatures. For example, during amplification bythermocycling or during denaturation of the double-stranded complexes,increase of temperature will result in the rapid evaporation of thedroplet. Therefore, the volume of the droplet can be monitored morefrequently and adjusting the droplet volume by re-hydration will be morefrequent. In the event of volume fluctuation such as loss of volume,sub-pico to nano volumes of water or solvent can be dispensed onto thedroplet or to the discrete feature comprising the droplet. Water orsolvent volumes of about 0.5 pL, 1 pL, 10 pL, of about 100 pL, of about1 nL, of about 10 nL, of about 100 nL can be dispensed this way. Wateror solvent volumes may be delivered by any conventional delivery meansas long that the volumes are controlled and accurate. In a preferredembodiment, water or solvent is dispensed using an inkjet device. Forexample, a typical inkjet printer is capable of producing 1.5 to 10 pLdroplets, while other commercial ultrasonic dispensing techniques canproduce 0.6 pL droplets. In some embodiments, water is added in a rapidseries of droplets. In some embodiments, water is dispensed whenregistering a loss of volume of more than 10%, of more than 25%, of morethan 35%, of more than 50%.

In other embodiment, evaporation rate is limited by raising the vaporrate or humidity surrounding the droplet. This can be performed, forexample, by placing “sacrificial” droplets around or in close proximityto the droplet of interest (e.g. droplet comprising theoligonucleotides) (see for example, Berthier E. et al., Lab Chip, 2008,8(6):852-859). In some embodiments, the surface or solid support isenclosed in a closed container to limit the evaporation.

Yet in another embodiment, the evaporation rate can be limited by addinga compound having a high boiling point component to the droplet(s) ofinterest, provided that the presence of the compound does not inhibitthe enzymatic reactions performed on the substrate. The boiling point ofa liquid is the temperature at which the liquid and vapor phases are inequilibrium with each other at a specified pressure. When heat isapplied to a solution, the temperature of the solution rises until thevapor pressure of the liquid equals the pressure of the surroundinggases. At this point, vaporization or evaporation occurs at the surfaceof the solution. By adding a high boiling point liquid to the droplet ofinterest, evaporation of the water content of a droplet may besubstantially reduced (see U.S. Pat. No. 6,177,558). In some embodiment,the high boiling point solution is a solvent. In some embodiments, thehigh boiling point liquid has a boiling point of at least 100° C., atleast 150° C., at least 200° C. In some embodiments, glycerol is addedto the solution, increasing the boiling point. Accordingly, the solutioncontaining the high boiling point liquid will evaporate at a much slowerrate at room temperature or at reaction conditions such as underthermocycling, extension, ligation and denaturation conditions.

Aspects of the invention provide methods for the amplification of one ormore single-stranded oligonucleotide on the support. Oligonucleotidesmay be amplified before or after being detached from the support and/oreluted in a droplet. Preferably, the oligonucleotides are amplified onthe solid support. One skilled in the art will appreciate thatoligonucleotides that are synthesized on solid support will comprise aphosphorylated 3′ end or an additional 3′-terminal nucleoside (e.g. T).The 3′-phosphorylated oligonucleotides are not suitable forpolynucleotide assembly as the oligonucleotides cannot be extended bypolymerase. In preferred aspects of the invention, the oligonucleotidesare first amplified and the amplified products are assembled into apolynucleotide. Accordingly, aspect of the invention provides methodswherein a set or subset of oligonucleotides, that are attached to at aset of subset of features of the support, are amplified by locallydelivering sub-microvolumes at addressable discrete features The term“amplification” means that the number of copies of a nucleic acidfragment is increased. As noted above, the oligonucleotides may be firstsynthesized onto discrete features of the surface, may be deposited onthe substrate or may be deposited on the substrate attached tonanoparticles. In a preferred embodiment, the oligonucleotides arecovalently attached to the surface or to nanoparticles deposited on thesurface. In an exemplary embodiment, locations or features comprisingthe oligonucleotides to be amplified are first selected. In a preferredembodiment, the selected features are in close proximity to each otherson the support. Aqueous or solvent solution is then deposited on theselected feature thereby forming a droplet comprising hydratedoligonucleotides. One would appreciate that each droplet is separatedfrom the other by surface tension. In some embodiment the solution canbe water, buffer or a solution promoting enzymatic reactions. In anexemplary embodiment, the solution includes, but is not limited to, asolution promoting primer extension. For example the solution may becomposed of oligonucleotides primer(s), nucleotides (dNTPs), buffer,polymerase and cofactors. In other embodiments, the solution is analkaline denaturing solution. Yet, in other embodiments, the solutionmay comprise oligonucleotides such as complementary oligonucleotides.

In some embodiments, oligonucleotides or polynucleotides are amplifiedwithin the droplet by solid phase PCR thereby eluting the amplifiedsequences into the droplet. In other embodiments, oligonucleotides orpolynucleotides are first detached form the solid support and thenamplified. For example, covalently-attached oligonucleotides aretranslated into surface supported DNA molecules through a process ofgaseous cleavage using amine gas.

Oligonucleotides can be cleaved with ammonia, or other amines, in thegas phase whereby the reagent gas comes into contact with theoligonucleotide while attached to, or in proximity to, the solid support(see Boal et al., NAR, 1996 (24(15):3115-7), U.S. Pat. Nos. 5,514,789;5,738,829 and 6,664,388). In this process, the covalent bond attachingthe oligonucleotides to the solid support is cleaved by exposing thesolid support to the amine gas under elevated pressure and/ortemperature. In some embodiments, this process may be used to “thin” thedensity of oligonucleotides at specific features. One skilled in the artwill appreciate that DNA microarrays can have very high density ofoligonucleotides on the surface (approximately 10⁸ molecules perfeature), which can generate steric hindrance to polymerases needed forPCR. Theoretically, the oligonucleotides are generally spaced apart byabout 2 nm to about 6 nm. For polymerases, a typical 6-subunit enzymecan have a diameter of about 12 nm. Therefore the support may need to becustom treated to address the surface density issue such that thespacing of surface-attached oligonucleotides can accommodate thephysical dimension of the enzyme. For example, a subset of theoligonucleotides can be chemically or enzymatically cleaved, orphysically removed from the microarray. Other methods can also be usedto modify the oligonucleotides such that when primers are applied andannealed to the oligonucleotides, at least some 3′ hydroxyl groups ofthe primers (start of DNA synthesis) are accessible by polymerase. Thenumber of accessible 3′ hydroxyl groups per spot can be stochastic orfixed. For example, the primers, once annealed, can be treated to removesome active 3′ hydroxyl groups, leaving a stochastic number of 3′hydroxyl groups that can be subject to chain extension reactions. Inanother example, a large linker molecule (e.g., a concatamer) can beused such that one and only one start of synthesis is available perspot, or in a subset of the oligonucleotides per spot.

The term “primer” as used herein refers to an oligonucleotide, eitherRNA or DNA, either single-stranded or double-stranded, either derivedfrom a biological system, generated by restriction enzyme digestion, orproduced synthetically which, when placed in the proper environment, isable to functionally act as an initiator of template-dependent nucleicacid synthesis. When presented with an appropriate nucleic acidtemplate, suitable nucleoside triphosphate precursors of nucleic acids,a polymerase enzyme, suitable cofactors and conditions such as asuitable temperature and pH, the primer may be extended at its 3′terminus by the addition of nucleotides by the action of a polymerase orsimilar activity to yield an primer extension product. The primer mayvary in length depending on the particular conditions and requirement ofthe application. For example, in diagnostic applications, theoligonucleotide primer is typically 15-25 or more nucleotides in length.The primer must be of sufficient complementarity to the desired templateto prime the synthesis of the desired extension product, that is, to beable anneal with the desired template strand in a manner sufficient toprovide the 3′ hydroxyl moiety of the primer in appropriatejuxtaposition for use in the initiation of synthesis by a polymerase orsimilar enzyme. It is not required that the primer sequence representsan exact complement of the desired template. For example, anon-complementary nucleotide sequence may be attached to the 5′ end ofan otherwise complementary primer. Alternatively, non-complementarybases may be interspersed within the oligonucleotide primer sequence,provided that the primer sequence has sufficient complementarity withthe sequence of the desired template strand to functionally provide atemplate-primer complex for the synthesis of the extension product.

According to aspects of the invention, hydrated oligonucleotides can beamplified within the droplet, the droplet acting as a virtual reactionchamber. In some embodiments, the entire support or array containing thediscrete features is subjected to amplification. In other embodiments,one or more selected discrete features are subjected to amplification.

Amplification of selected independent features (being separated fromeach others) can be performed by locally heating at least one discretefeature. Discrete features may be locally heated by any means known inthe art. For example, the discrete features may be locally heated usinga laser source of energy that can be controlled in a precise x-ydimension thereby individually modulating the temperature of a droplet.In another example, the combination of a broader beam laser with a maskcan be used to irradiate specific features. In some embodiments, methodsto control temperature on the support so that enzymatic reactions cantake place on a support (PCR, ligation or any other temperaturesensitive reaction) are provided. In some embodiments, a scanning laseris used to control the thermocycling on distinct features on the solidsupport. The wavelength used can be chosen from wide spectrum (100 nm to100,000 nm, i.e. from ultraviolet to infrared). In some embodiments, thefeature on which the droplet is spotted comprises an optical absorber orindicator. In some other embodiment, optical absorbent material can beadded on the surface of the droplet. In some embodiments, the solidsupport is cooled by circulation of air or fluid. The energy to bedeposited can be calculated based on the absorbance behavior. In someembodiments, the temperature of the droplet can be modeled usingthermodynamics. The temperature can be measured by an LCD like materialor any other in-situ technology. Yet in another embodiment, the wholesupport can be heated and cooled down to allow enzymatic reactions totake place. One method to control the temperature of the surfacedroplets is by using a scanning optical energy deposition setup. Anenergy source can be directed by a scanning setup to deposit energy atvarious locations on the surface of the solid support comprisingattached or supported molecules. Optical absorbent material can be addedon the surface of the solid support or on the surface of droplet.Optical energy source, such as a high intensity lamp, laser, or otherelectromagnetic energy source (including microwave) can be used. Thetemperature of the different reaction sites can be controlledindependently by controlling the energy deposited at each of thefeatures.

For example, a Digital Micromirror Device (DMD) can be used fortemperature control. DMD is an optical semiconductor. See, for example,U.S. Pat. No. 7,498,176. In some embodiments, a DMD can be used toprecisely heat selected features or droplets on the solid support. TheDMD can be a chip having on its surface several hundred thousandmicroscopic mirrors arranged in a rectangular array which correspond tothe features or droplets to be heated. The mirrors can be individuallyrotated (e.g., ±10-12°), to an on or off state. In the on state, lightfrom a light source (e.g., a bulb) is reflected onto the solid supportto heat the selected spots or droplets. In the off state, the light isdirected elsewhere (e.g., onto a heatsink). In one example, the DMD canconsist of a 1024×768 array of 16 μm wide micromirrors. These mirrorscan be individually addressable and can be used to create any givenpattern or arrangement in heating different features on the solidsupport. The features can also be heated to different temperatures,e.g., by providing different wavelength for individual spots, and/orcontrolling time of irradiation.

One would appreciate that amplification occurs only on featurescomprising hydrated template oligonucleotides (i.e. local amplificationat features comprising a droplet). Different set of features may beamplified in a parallel or sequential fashion with parallel orsequential rounds of hydrating (i.e. dispensing a droplet on a specificfeature), amplifying oligonucleotides and drying the set of features. Insome embodiments, the support is dried by evaporating liquid in a vacuumwhile heating. Thus, after each round of amplification, the support willcomprise a set of droplets containing oligonucleotides duplexes. Thecomplementary oligonucleotides can be released in solution within thedroplet and be collected. Alternatively, complementary oligonucleotidesmay be dried onto the discrete features for storage or furtherprocessing. Yet, complementary oligonucleotides can be subjected tofurther reactions such as error filtration or assembly. In someembodiments, a different set or subset of features can then be hydratedand a different set or subset of template oligonucleotides can beamplified as described herein. For example, a droplet can be dispensed(e.g., inkjetted) on a support. The droplet can contain various reagentssuch as enzymes, buffers, dNTPs, primers, etc. The droplet covers adiscrete feature (a feature corresponds to a predefined sequence) on thesupport. PCR can be carried out to synthesize oligonucleotidescomplementary to template oligonucleotides that are attached to thefeature. In the case of the enzymatic amplification, the solutionincludes but is not limited to primers, nucleotides, buffers, cofactors,and enzyme. For example, an amplification reaction includes DNApolymerase, nucleotides (e.g. dATP, dCTP, dTTP, dGTP), primers andbuffer.

In some embodiments, the oligonucleotides may comprise universal (commonto all oligonucleotides), semiuniversal (common to at least of portionof the oligonucleotides) or individual or unique primer (specific toeach oligonucleotide) binding sites on either the 5′ end or the 3′ endor both. As used herein, the term “universal” primer or primer bindingsite means that a sequence used to amplify the oligonucleotide is commonto all oligonucleotides such that all such oligonucleotides can beamplified using a single set of universal primers. In othercircumstances, an oligonucleotide contains a unique primer binding site.As used herein, the term “unique primer binding site” refers to a set ofprimer recognition sequences that selectively amplifies a subset ofoligonucleotides. In yet other circumstances, an oligonucleotidecontains both universal and unique amplification sequences, which canoptionally be used sequentially.

In some embodiments, primers/primer binding site may be designed to betemporary. For example, temporary primers may be removed by chemical,light based or enzymatic cleavage. For example, primers/primer bindingsites may be designed to include a restriction endonuclease cleavagesite. In an exemplary embodiment, a primer/primer binding site containsa binding and/or cleavage site for a type IIs restriction endonuclease.In such case, amplification sequences may be designed so that once adesired set of oligonucleotides is amplified to a sufficient amount, itcan then be cleaved by the use of an appropriate type IIs restrictionenzyme that recognizes an internal type IIs restriction enzyme sequenceof the oligonucleotide. In some embodiments, after amplification, thepool of nucleic acids may be contacted with one or more endonucleases toproduce double-stranded breaks thereby removing the primers/primerbinding sites. In certain embodiments, the forward and reverse primersmay be removed by the same or different restriction endonucleases. Anytype of restriction endonuclease may be used to remove theprimers/primer binding sites from nucleic acid sequences. A wide varietyof restriction endonucleases having specific binding and/or cleavagesites are commercially available, for example, from New England Biolabs(Beverly, Mass.). In various embodiments, restriction endonucleases thatproduce 3′ overhangs, 5′ overhangs or blunt ends may be used. When usinga restriction endonuclease that produces an overhang, an exonuclease(e.g., RecJ_(f), Exonuclease I, Exonuclease T, S₁ nuclease, P₁ nuclease,mung bean nuclease, T4 DNA polymerase, CEL I nuclease, etc.) may be usedto produce blunt ends. Alternatively, the sticky ends formed by thespecific restriction endonuclease may be used to facilitate assembly ofsubassemblies in a desired arrangement. In an exemplary embodiment, aprimer/primer binding site that contains a binding and/or cleavage sitefor a type IIs restriction endonuclease may be used to remove thetemporary primer. The term “type-IIs restriction endonuclease” refers toa restriction endonuclease having a non-palindromic recognition sequenceand a cleavage site that occurs outside of the recognition site (e.g.,from 0 to about 20 nucleotides distal to the recognition site). Type IIsrestriction endonucleases may create a nick in a double-stranded nucleicacid molecule or may create a double-stranded break that produces eitherblunt or sticky ends (e.g., either 5′ or 3′ overhangs). Examples of TypeIIs endonucleases include, for example, enzymes that produce a 3′overhang, such as, for example, Bsr I, Bsm I, BstF5 I, BsrD I, Bts I,Mnl I, BciV I, Hph I, Mbo II, Eci I, Acu I, Bpm I, Mme I, BsaX I, Bcg I,Bae I, Bfi I, TspDT I, TspGW I, Taq II, Eco57 I, Eco57M I, Gsu I, Ppi I,and Psr I; enzymes that produce a 5′ overhang such as, for example, BsmAI, Ple I, Fau I, Sap I, BspM I, SfaN I, Hga I, Bvb I, Fok I, BceA I,BsmF I, Ksp632 I, Eco31 I, Esp3 I, Aar I; and enzymes that produce ablunt end, such as, for example, Mly I and Btr I. Type-IIs endonucleasesare commercially available and are well known in the art (New EnglandBiolabs, Beverly, Mass.).

In some embodiments, the primer is a primer containing multiple uracil(U). The primer is first annealed to a support-bound single-strandedoligonucleotide and extended with the addition of dNTPs and anappropriate polymerase under appropriate conditions and temperature. Ina subsequent step, the primer is removed. In some embodiments, uracilDNA glycosylase (UDG) may be used to hydrolyze a uracil-glycosidic bondin a nucleic acid thereby removing uracil and creating analkali-sensitive a basic site in the DNA which can be subsequentlyhydrolyzed by endonuclease, heat or alkali treatment. As a result, aportion of one strand of a double-stranded nucleic acid may be removedthereby exposing the complementary sequence in the form of asingle-stranded overhang. This approach requires the deliberateincorporation of one or more uracil bases on one strand of adouble-stranded nucleic acid fragment. This may be accomplished, forexample, by amplifying a nucleic acid fragment using an amplificationprimer that contains a 3′ terminal uracil. After treatment with UDG, theregion of the primer 5′ to the uracil may be released (e.g., upondilution, incubation, exposure to mild denaturing conditions, etc.)thereby exposing the complementary sequence as a single-strandedoverhang. It should be appreciated that the length of the overhang maybe determined by the position of the uracil on the amplifying primer andby the length of the amplifying primer. In some embodiments, mixture ofUracil DNA glycosylase (UDG) and the DNA glycosylase-lyase EndonucleaseVIII, such as USER™ (Uracil-Specific Excision Reagent) is used. UDGcatalyses the excision of a uracil base, forming an abasic site whileleaving the phosphodiester backbone intact. The lyase activity ofEndonuclease VIII breaks the phosphodiester backbone at the 3′ and 5′sides of the abasic site so that base-free deoxyribose is released.

After amplification, the polymerase may be deactivated to preventinterference with the subsequent steps. A heating step (e.g. hightemperature) can denature and deactivate most enzymes which are notthermally stable. Enzymes may be deactivated in presence (e.g. withinthe droplet) or in the absence of liquid (e.g. dry array). Heatdeactivation on a dry support has the advantage to deactivate theenzymes without any detrimental effect on the oligonucleotides. In someembodiments, a non-thermal stable version of the thermally stable PCRDNA Polymerase may be used, although the enzyme is less optimized forerror rate and speed. Alternatively, Epoxy dATP can be use to inactivatethe enzyme.

In some embodiments, discrete features may contain oligonucleotides thatare substantially complementary (e.g. 50%, 60%, 70%, 80%, 90%, 95%, 98%,99% or 100%). Template oligonucleotides can have inherent errors as theyare generally chemically synthesized (e.g., deletions at a rate of 1 in100 bases and mismatches and insertions at about 1 in 400 bases).Assuming an average error rate of 1 in 300 bases and an average templateoligonucleotide size of 70 bases, every 1 in 4 template oligonucleotideswill contain an error compared to a reference sequence (e.g., thewide-type sequence of a gene of interest). For example, a templateoligonucleotide can contain an error which can be a mismatch, deletion,or insertion. In PCR synthesis, the error is retained in the synthesizedoligonucleotide. Additional errors can be introduced during PCRreactions. Accordingly, methods for error correction are needed forhigh-fidelity gene synthesis/assembly.

Accordingly, some aspects of the invention relate to the recognition andlocal removal of double-stranded oligonucleotides containing sequencemismatch errors at specific features. In one preferred embodiment of theinvention, mismatch recognition can be used to control the errorsgenerated during oligonucleotide synthesis, gene assembly, and theconstruction of longer polynucleotides. After amplification, thetotality of the features or a set of the features comprisingoligonucleotide duplexes are first subjected to round(s) of melting andannealing as described above. In a preferred aspect of the invention,oligonucleotides having predefined sequences are assembled after beingamplified and error-filtered. In some embodiments, two adjacent dropletscontaining two multiple copies of different oligonucleotides orpolynucleotides in solution are combined by merging the appropriatedroplets on the solid support. The solid support comprises different andunique molecules supported or attached to the surface, a unique moleculesupported or attached to the surface at multiple positions other uniquemolecules supported or attached to the surface. On the solid supportsurface an existing pattern of molecules can be found. Differentmolecules or oligonucleotides can exist at different positions. Oneshould appreciate that the arrangement of these unique molecules can bedesigned to strategically allow the subsequent combining of the contentsof these sites. For example, these unique molecules can be arranged in achecker board pattern.

Aspect of the invention also relate to methods to shuffle moleculeswithin one or more features of a high diversity library on a solidsupport. Various fluidic methods can be used to achieve shuffling. Insome embodiments, shuffling can be performed with depositedmicro-volumes or without micro-volumes. For example, with reference toFIG. 5, shuffling can be implemented in individual micro-volumes (502)created at the deposition of one or more fluids. Members of the highdiversity library (504) can be immobilized on a surface (501). Shufflingcan be achieved by heating and cooling the micro-volumes (502), causinglocalized melting and reannealing at each location where a member of thelibrary is positioned (FIG. 5A). These micro-volumes can be created in aserial manner as illustrated in FIG. 5B showing a process progressingfrom right to left. For each member of the library, first, a liquidsuitable for the shuffling reaction is deposited (505), resulting inmicro-volumes (502); following the deposition of (505), a second fluid(506) that is immiscible to (505) is deposited over the micro-volumes(502), forming a blanket (503) over the micro-volumes (502). Thisprocess can be carried out until all the desirable members of the highdiversity library (504) are covered by micro-volume (502) and/or blanket(503).

In another example, the surface properties of the substrate (501) can beutilized to achieve the structure in FIG. 5A on a global scale. Asillustrated in FIG. 5C, the substrate (501) starts fully immersed in afluid (511) contained in a container (510), such that all the members ofthe high diversity library are in contact with the fluid (511), as shownin FIG. 5C1. The fluid (511) is such that it has low contact angle withthe substrate surface where the members of the library resides. A secondfluid (512) is added to the container (510) which is immiscible to thefirst fluid (511), forming an interface (513). The second fluid (512) issuch that it has a different contact angle (preferably higher) with thesubstrate surface where the members of the library resides. Yet, betweenthe members of the library, the second fluid (512) may have a lowercontact angle to the surface. When the substrate is moved from beingfully immersed in (511) through the interface (513), small volumes ofthe first fluid (511) is retained on the surface of the substrate (510)where the members of the high diversity library resides, formingmicro-volumes (504). Furthermore, once the surface passes the interface(513), the micro-volumes are covered by a blanket formed by fluid (512).The resulting structure of the micro-volumes is shown in FIG. 5C2.

The process illustrated in FIG. 5C can be carried out without thecontainer (510) by using an arrangement shown in FIG. 5D. Two meniscuscapillary heads (520, 512) are lowered to near contact with thesubstrate (501). Fluids (511) and (512) are supplied into the meniscuscapillary heads (520, 521) to form a small volume (522, 523) thatcontacts both the meniscus capillary heads (520, 521) and the substrate(501). The relative motion of the heads and the substrate is shown inthe direction of the arrow. The volumes (522, 523) may be placed closeenough to form an interface, or separated enough to from a space betweenthe volumes. Micro-volumes are created as the members of the library(504) passes under first the reaction fluid meniscus (522) then theblanket fluid meniscus (523), resulting in the encapsulated features asshown in FIG. 5A.

Shuffling can also be achieved without micro-volumes. In one example, asoutlined in FIG. 6, the precise control of short periods of time forwhich temperature of the substrate (601) and fluid (600) is raised tocause melting is used to achieve localized melting and reannealingthrough diffusion limit. FIG. 6A1 shows the setting prior to melting,where homoduplexes (602, 603) are attached to the surface via thecovalent bond of the template strand. When temperature is raised toabove the melting temperature of the homo-duplexes, melting takes placeand the single stranded copies (604, 605) are released into the fluid(600). FIG. 6B1 shows the melting at a time immediately after thetemperature increases, and FIG. 6C1 shows the melting at a later timethan that of FIG. 6B1, and FIG. 6D1 shows the melting at a later timethan that of FIG. 6C1. The melted single strand copies (604, 605)diffuse away from their original location and the diffusion is governedby the diffusion equation. FIGS. 6A2, 6B2, 6C2, and 6D2 are intended toillustrate the diffusion process as time progresses by showing theconcentration of duplexes (e.g., signal strength) at two adjacentpositions on the substrate (601). The longer the time is allowed fordiffusion, the further the molecules would travel. The characteristicdiffusion distance (Ld) is related to the diffusion coefficient (D) andtime (t):

Ld=sqrt(4Dt)

The time that is allowed for diffusion can be controlled, and thus thecharacteristic diffusion distance can be controlled also, since thediffusion coefficient is determined by the molecular species. As shownin FIG. 6E, when reannealing is induced (by lowering temperature) aftera brief period of melting time, the reannealed heteroduplexes arerecaptured back to the original positions (606, 607), achieving ashuffling operation. If the melting time is allowed to proceed longer,more of the molecules (608) will diffuse away into the bulk of the fluid(600) and will be less likely to be recaptured back to the originalposition.

In some embodiments, shuffling can be achieved with a droplet that isactively evaporating. In the example shown in FIGS. 7A-7E, the surface(701) is first raised to a temperature above the melting temperature ofthe homo-duplexes formed by the template (702) and copies (703, FIG.7A). A volume (704) is deposited to cover the homo-duplexes (FIG. 7B),and since the surface is already at the high melting temperature, thecopies are melted (706) into the fluid (705) which is reducing in volumerapidly due to evaporation (FIG. 7C). The droplet is allowed tocompletely dry, depositing the copies (707) onto the surface (FIG. 7D).The substrate is then cooled to below the melting temperature of theparticular population, and rehydrated to resuspend the copies (707) intothe fluid volume (708, FIG. 7E). Subsequent reannealing of the copies(707) to the templates (702) results in a shuffled heteroduplex group,completing the shuffling operation.

Following melting and reannealing, heteroduplexes can be removed byvarious methods described above. The remaining duplexes can then besubject to melting again to denature some or all of the duplexes. Insome embodiments, some duplexes (e.g., the cleaved or truncatedduplexes) are selectively melted, leaving others (e.g., the full-lengthduplexes) intact. The conditions for such stringent melt (e.g., aprecise melting temperature) can be determined by observing a real-timemelt curve. In an exemplary melt curve analysis, PCR products are slowlyheated in the presence of double-stranded DNA (dsDNA) specificfluorescent dyes (e.g., SYBR Green, LCGreen, SYTO9 or EvaGreen). Withincreasing temperature the dsDNA denatures (melts), releasing thefluorescent dye with a resultant decrease in the fluorescent signal. Thetemperature at which dsDNA melts is determined by factors such asnucleotide sequence, DNA length and GC/AT ratio. Typically, G-C basepairs in a duplex are estimated to contribute about 3° C. to the Tm,while A-T base pairs are estimated to contribute about 2° C., up to atheoretical maximum of about 80-100° C. However, more sophisticatedmodels of Tm are available and may be in which G-C stackinginteractions, solvent effects, the desired assay temperature and thelike are taken into account. Melt curve analysis can detect a singlebase difference. Various methods for accurate temperature control atindividual features can be used as disclosed above.

Some aspects of the invention relate to destination selection androuting of the isolated volumes and therefore to the control of thelocation or footprint of merged volumes. One would appreciate that asindividual regions of the support are addressable, individual isolatedvolumes such as droplets may be controlled individually. In someembodiments, it is preferable to place isolated volumes onto adjacentregions or features to allow merging of the volumes. Yet, in otherembodiments, isolated volumes are directed or routed to a selecteddestination. In some embodiments, the substrate of the support issubstantially planar and droplets are routed using a two-dimensionalpath (e.g. x,y axis). Droplets may be moved to bring them to selectedlocations for further processing, to be merged with a second isolatedvolume into a second stage droplet at preselected locations and/orduring the transport, to remove some reactants from the droplet(referred as. “wash-in-transport” process, as described herein) etc.

In some embodiments, step-wise hierarchical and/or sequential assemblycan be used to assemble oligonucleotides and longer polynucleotides. Ina preferred embodiment, the methods use hierarchical assembly of two ormore oligonucleotides or two or more subassemblies polynucleotidefragments at a time. Neighboring droplets can be manipulated (moveand/or merged, as described above) to merge following a hierarchicalstrategy thereby improving assembly efficiency. In some embodiments,each droplet contains oligonucleotides with predefined and differentnucleic acid sequences. In some embodiments, two droplets are movedfollowing a predefined path to an oligonucleotide-free position. In apreferred embodiment, the assembly molecules (e.g. oligonucleotides) arepre-arranged on the support surface at pre-determined discrete features.One should appreciate that isolated volumes may be routed independentlyin a sequential or highly parallel fashion. Droplets may be routed usingelectrowetting-based techniques (see for example, U.S. Pat. No.6,911,132 and U.S. Patent Application 2006/0054503). Electrowettingprinciple is based on manipulating droplets on a surface comprising anarray of electrodes and using voltage to change the interfacial tension.In some embodiments, droplets are moved using a wettability gradient. Ithas been shown that droplets placed on wettability gradient surfacestypically move in the direction of increasing wettability (see Zielkeand Szymczyk, Eur. Phys. J. Special Topics, 166, 155-158 (2009)). Inother embodiments, droplets may be moved using a thermal gradient. Whenplaced on a thermal gradient, droplets move from higher temperaturelocations towards lower temperature locations. Moving droplets usingelectrowetting, temperature gradients and wettability gradients dependon the liquid (e.g. aqueous, non-aqueous, solute concentration), thesize of the droplets and/or the steepness of the gradient.

Another benefit of the move operation described herein is theimplementation of a “wash” operation. The movement of the liquid awayfrom a surface feature allows the separation of the surface-boundmolecules (e.g. oligonucleotides) from the molecules in solution. Hence,a wash operation is therefore implemented. For example,wash-in-transportation can be used to remove the templateoligonucleotides form the complementary oligonucleotides afteramplification. In some embodiments, “wash-in transportation” features orwash spots may be placed adjacent to features where oligonucleotideprocessing takes place. Wash spots can be placed adjacent to thefeatures. Undesirable products released in the droplet solution on thefeatures can be moved to the wash spots respectively. In someembodiments, the support provides one wash spot for each assemblyfeature or a common wash spot for two or more assembly features.Wash-in-transportation process can also be used to remove unwantederror-containing oligonucleotides from stable duplexes after annealingand stringent melt. For example, stringent melt features can be placedalong the path of assembly progression, allowing for stringent melterror correction as described above. Similarly, the support may compriseone SM spot for each assembly step or a common SM spot for two or moreassembly features.

In some embodiments, the content of two microvolumes such as dropletsare merged to allow for polynucleotide assembly. For example, two firststage droplets can be merged forming a larger second stage droplet. Insome embodiments, “merger” droplets or “anchor” droplets are added whichmay contain or not contain enzyme (e.g. polymerase, ligase, etc.),additional oligonucleotides and all reagents to allow assembly by PCR orby ligation (enzymatic or chemical) or by any combination of enzymaticreaction. For example, oligonucleotides in a given droplet may hybridizeto each other and may assemble by PCR or ligation. The merged dropletsor second stage droplets contain polynucleotides subassemblies and canbe subsequently merged to form larger droplets or third stage dropletcontaining larger fragments. As used herein the term subassembly refersto a nucleic acid molecule that has been assembled from a set ofoligonucleotides. Preferably, a subassembly is at least 2-fold or morelong than the oligonucleotides. For example, a subassembly may be about100, 200, 300, 400, 500, 600, or ore bases long. One should appreciatethat the use of droplets as isolated reaction volume enables a highlyparallel system. In some embodiments, at least 100, at least 1,000reactions can take place in parallel. In some embodiments, the primersare immobilized on the support in close proximity to the spotscontaining the oligonucleotides to be assembled. In some embodiments,the primers are cleaved in situ. In some embodiments, the primers aresupported on the solid support. The primers may then be cleaved in situand eluted within a droplet that will subsequently merged with a dropletcontaining solid supported or eluted oligonucleotides.

In certain embodiments, the oligonucleotides are designed to provide thefull sense (plus strand) and antisense (minus strand) strands of thepolynucleotide construct. After hybridization of the plus and minusstrand oligonucleotides, two double-stranded oligonucleotides aresubjected to ligation in order to form a first subassembly product.Subassembly products are then subjected to ligation to form a largernucleic acid or the full nucleic acid sequence.

Ligase-based assembly techniques may involve one or more suitable ligaseenzymes that can catalyze the covalent linking of adjacent 3′ and 5′nucleic acid termini (e.g., a 5′ phosphate and a 3′ hydroxyl of nucleicacid(s) annealed on a complementary template nucleic acid such that the3′ terminus is immediately adjacent to the 5′ terminus). Accordingly, aligase may catalyze a ligation reaction between the 5′ phosphate of afirst nucleic acid to the 3′ hydroxyl of a second nucleic acid if thefirst and second nucleic acids are annealed next to each other on atemplate nucleic acid). A ligase may be obtained from recombinant ornatural sources. A ligase may be a heat-stable ligase. In someembodiments, a thermostable ligase from a thermophilic organism may beused. Examples of thermostable DNA ligases include, but are not limitedto: Tth DNA ligase (from Thermus thermophilus, available from, forexample, Eurogentec and GeneCraft); Pfu DNA ligase (a hyperthermophilicligase from Pyrococcus furiosus); Taq ligase (from Thermus aquaticus),any other suitable heat-stable ligase, or any combination thereof. Insome embodiments, one or more lower temperature ligases may be used(e.g., T4 DNA ligase). A lower temperature ligase may be useful forshorter overhangs (e.g., about 3, about 4, about 5, or about 6 baseoverhangs) that may not be stable at higher temperatures.

Non-enzymatic techniques can be used to ligate nucleic acids. Forexample, a 5′-end (e.g., the 5′ phosphate group) and a 3′-end (e.g., the3′ hydroxyl) of one or more nucleic acids may be covalently linkedtogether without using enzymes (e.g., without using a ligase). In someembodiments, non-enzymatic techniques may offer certain advantages overenzyme-based ligations. For example, non-enzymatic techniques may have ahigh tolerance of non-natural nucleotide analogues in nucleic acidsubstrates, may be used to ligate short nucleic acid substrates, may beused to ligate RNA substrates, and/or may be cheaper and/or more suitedto certain automated (e.g., high throughput) applications.

Non-enzymatic ligation may involve a chemical ligation. In someembodiments, nucleic acid termini of two or more different nucleic acidsmay be chemically ligated. In some embodiments, nucleic acid termini ofa single nucleic acid may be chemically ligated (e.g., to circularizethe nucleic acid). It should be appreciated that both strands at a firstdouble-stranded nucleic acid terminus may be chemically ligated to bothstrands at a second double-stranded nucleic acid terminus. However, insome embodiments only one strand of a first nucleic acid terminus may bechemically ligated to a single strand of a second nucleic acid terminus.For example, the 5′ end of one strand of a first nucleic acid terminusmay be ligated to the 3′ end of one strand of a second nucleic acidterminus without the ends of the complementary strands being chemicallyligated.

Accordingly, a chemical ligation may be used to form a covalent linkagebetween a 5′ terminus of a first nucleic acid end and a 3′ terminus of asecond nucleic acid end, wherein the first and second nucleic acid endsmay be ends of a single nucleic acid or ends of separate nucleic acids.In one aspect, chemical ligation may involve at least one nucleic acidsubstrate having a modified end (e.g., a modified 5′ and/or 3′ terminus)including one or more chemically reactive moieties that facilitate orpromote linkage formation. In some embodiments, chemical ligation occurswhen one or more nucleic acid termini are brought together in closeproximity (e.g., when the termini are brought together due to annealingbetween complementary nucleic acid sequences). Accordingly, annealingbetween complementary 3′ or 5′ overhangs (e.g., overhangs generated byrestriction enzyme cleavage of a double-stranded nucleic acid) orbetween any combination of complementary nucleic acids that results in a3′ terminus being brought into close proximity with a 5′ terminus (e.g.,the 3′ and 5′ termini are adjacent to each other when the nucleic acidsare annealed to a complementary template nucleic acid) may promote atemplate-directed chemical ligation. Examples of chemical reactions mayinclude, but are not limited to, condensation, reduction, and/orphoto-chemical ligation reactions. It should be appreciated that in someembodiments chemical ligation can be used to produce naturally occurringphosphodiester internucleotide linkages, non-naturally-occurringphosphamide pyrophosphate internucleotide linkages, and/or othernon-naturally-occurring internucleotide linkages.

In some embodiments, the process of chemical ligation may involve one ormore coupling agents to catalyze the ligation reaction. A coupling agentmay promote a ligation reaction between reactive groups in adjacentnucleic acids (e.g., between a 5′-reactive moiety and a 3′-reactivemoiety at adjacent sites along a complementary template). In someembodiments, a coupling agent may be a reducing reagent (e.g.,ferricyanide), a condensing reagent such (e.g., cyanoimidazole, cyanogenbromide, carbodiimide, etc.), or irradiation (e.g., UV irradiation forphoto-ligation).

In some embodiments, a chemical ligation may be an autoligation reactionthat does not involve a separate coupling agent. In autoligation, thepresence of a reactive group on one or more nucleic acids may besufficient to catalyze a chemical ligation between nucleic acid terminiwithout the addition of a coupling agent (see, for example, Xu et al.,(1997) Tetrahedron Lett. 38:5595-8). Non-limiting examples of thesereagent-free ligation reactions may involve nucleophilic displacementsof sulfur on bromoacetyl, tosyl, or iodo-nucleoside groups (see, forexample, Xu et al., (2001) Nat. Biotech. 19:148-52). Nucleic acidscontaining reactive groups suitable for autoligation can be prepareddirectly on automated synthesizers (see, for example, Xu et al., (1999)Nuc. Acids Res. 27:875-81). In some embodiments, a phosphorothioate at a3′ terminus may react with a leaving group (such as tosylate or iodide)on a thymidine at an adjacent 5′ terminus. In some embodiments, twonucleic acid strands bound at adjacent sites on a complementary targetstrand may undergo auto-ligation by displacement of a 5′-end iodidemoiety (or tosylate) with a 3′-end sulfur moiety. Accordingly, in someembodiments the product of an autoligation may include anon-naturally-occurring internucleotide linkage (e.g., a single oxygenatom may be replaced with a sulfur atom in the ligated product).

In some embodiments, a synthetic nucleic acid duplex can be assembledvia chemical ligation in a one step reaction involving simultaneouschemical ligation of nucleic acids on both strands of the duplex. Forexample, a mixture of 5′-phosphorylated oligonucleotides correspondingto both strands of a target nucleic acid may be chemically ligated by a)exposure to heat (e.g., to 97° C.) and slow cooling to form a complex ofannealed oligonucleotides, and b) exposure to cyanogen bromide or anyother suitable coupling agent under conditions sufficient to chemicallyligate adjacent 3′ and 5′ ends in the nucleic acid complex.

In some embodiments, a synthetic nucleic acid duplex can be assembledvia chemical ligation in a two step reaction involving separate chemicalligations for the complementary strands of the duplex. For example, eachstrand of a target nucleic acid may be ligated in a separate reactioncontaining phosphorylated oligonucleotides corresponding to the strandthat is to be ligated and non-phosphorylated oligonucleotidescorresponding to the complementary strand. The non-phosphorylatedoligonucleotides may serve as a template for the phosphorylatedoligonucleotides during a chemical ligation (e.g., using cyanogenbromide). The resulting single-stranded ligated nucleic acid may bepurified and annealed to a complementary ligated single-stranded nucleicacid to form the target duplex nucleic acid (see, for example, Shabarovaet al., (1991) Nucl. Acids Res. 19:4247-51).

In one aspect, a nucleic acid fragment may be assembled in a polymerasemediated assembly reaction from a plurality of oligonucleotides that arecombined and extended in one or more rounds of polymerase-mediatedextensions. In some embodiments, the oligonucleotides are overlappingoligonucleotides covering the full sequence but leaving single-strandedgaps that may be filed in by chain extension. The plurality of differentoligonucleotides may provide either positive sequences (plus strand),negative sequences (minus strand), or a combination of both positive andnegative sequences corresponding to the entire sequence of the nucleicacid fragment to be assembled. In some embodiments, one or moredifferent oligonucleotides may have overlapping sequence regions (e.g.,overlapping 5′ regions or overlapping 3′ regions). Overlapping sequenceregions may be identical (i.e., corresponding to the same strand of thenucleic acid fragment) or complementary (i.e., corresponding tocomplementary strands of the nucleic acid fragment). The plurality ofoligonucleotides may include one or more oligonucleotide pairs withoverlapping identical sequence regions, one or more oligonucleotidepairs with overlapping complementary sequence regions, or a combinationthereof. Overlapping sequences may be of any suitable length. Forexample, overlapping sequences may encompass the entire length of one ormore nucleic acids used in an assembly reaction. Overlapping sequencesmay be between about 5 and about 500 oligonucleotides long (e.g.,between about 10 and 100, between about 10 and 75, between about 10 and50, about 20, about 25, about 30, about 35, about 45, about 50, etc.).However, shorter, longer, or intermediate overlapping lengths may beused. It should be appreciated that overlaps between different inputnucleic acids used in an assembly reaction may have different lengths.

Polymerase-based assembly techniques may involve one or more suitablepolymerase enzymes that can catalyze a template-based extension of anucleic acid in a 5′ to 3′ direction in the presence of suitablenucleotides and an annealed template. A polymerase may be thermostable.A polymerase may be obtained from recombinant or natural sources. Insome embodiments, a thermostable polymerase from a thermophilic organismmay be used. In some embodiments, a polymerase may include a 3′→5′exonuclease/proofreading activity. In some embodiments, a polymerase mayhave no, or little, proofreading activity (e.g., a polymerase may be arecombinant variant of a natural polymerase that has been modified toreduce its proofreading activity). Examples of thermostable DNApolymerases include, but are not limited to: Taq (a heat-stable DNApolymerase from the bacterium Thermus aquaticus); Pfu (a thermophilicDNA polymerase with a 3′→5′ exonuclease/proofreading activity fromPyrococcus furiosus, available from for example Promega); VentR® DNAPolymerase and VentRO (exo−) DNA Polymerase (thermophilic DNApolymerases with or without a 3′→5′ exonuclease/proofreading activityfrom Thermococcus litoralis; also known as Th polymerase); Deep VentR®DNA Polymerase and Deep VentR® (exo−) DNA Polymerase (thermophilic DNApolymerases with or without a 3′→5′ exonuclease/proofreading activityfrom Pyrococcus species GB-D; available from New England Biolabs); KODHiFi (a recombinant Thermococcus kodakaraensis KODI DNA polymerase witha 3′→5′ exonuclease/proofreading activity, available from Novagen,);BIO-X-ACT (a mix of polymerases that possesses 5′-3′ DNA polymeraseactivity and 3′→5′ proofreading activity); Klenow Fragment (anN-terminal truncation of E. coli DNA Polymerase I which retainspolymerase activity, but has lost the 5′→3′ exonuclease activity,available from, for example, Promega and NEB); Sequenase™ (T7 DNApolymerase deficient in T-5′ exonuclease activity); Phi29 (bacteriophage29 DNA polymerase, may be used for rolling circle amplification, forexample, in a TempliPhi™ DNA Sequencing Template Amplification Kit,available from Amersham Biosciences); TopoTaq (a hybrid polymerase thatcombines hyperstable DNA binding domains and the DNA unlinking activityof Methanopyrus topoisomerase, with no exonuclease activity, availablefrom Fidelity Systems); TopoTaq HiFi which incorporates a proofreadingdomain with exonuclease activity; Phusion™ (a Pyrococcus-like enzymewith a processivity-enhancing domain, available from New EnglandBiolabs); any other suitable DNA polymerase, or any combination of twoor more thereof.

In some embodiments, the polymerase can be a SDP (strand-displacingpolymerase; e.g, an SDPe− which is an SDP with no exonuclease activity).This allows isothermal PCR (isothermal extension, isothermalamplification) at a uniform temperature. As the polymerase (for example,Phi29, Bst) travels along a template it displaces the complementarystrand (e.g., created in previous extension reactions). As the displacedDNAs are single-stranded, primers can bind at a consistent temperature,removing the need for any thermocycling during amplification, therebyavoiding or decreasing evaporation of the reaction mixture.

It should be appreciated that the description of the assembly reactionsin the context of the oligonucleotides is not intended to be limiting.For example, other polynucleotides (e.g. single-stranded,double-stranded polynucleotides, restriction fragments, amplificationproducts, naturally occurring polynucleotides, etc.) may be included inan assembly reaction, along with one or more oligonucleotides, in orderto generate a polynucleotide of interest.

Aspects of the methods and devices provided herein may includeautomating one or more acts described herein. In some embodiments, oneor more steps of an amplification and/or assembly reaction may beautomated using one or more automated sample handling devices (e.g., oneor more automated liquid or fluid handling devices). Automated devicesand procedures may be used to deliver reaction reagents, including oneor more of the following: starting nucleic acids, buffers, enzymes(e.g., one or more ligases and/or polymerases), nucleotides, salts, andany other suitable agents such as stabilizing agents. Automated devicesand procedures also may be used to control the reaction conditions. Forexample, an automated thermal cycler may be used to control reactiontemperatures and any temperature cycles that may be used. In someembodiments, a scanning laser may be automated to provide one or morereaction temperatures or temperature cycles suitable for incubatingpolynucleotides. Similarly, subsequent analysis of assembledpolynucleotide products may be automated. For example, sequencing may beautomated using a sequencing device and automated sequencing protocols.Additional steps (e.g., amplification, cloning, etc.) also may beautomated using one or more appropriate devices and related protocols.It should be appreciated that one or more of the device or devicecomponents described herein may be combined in a system (e.g., a roboticsystem) or in a micro-environment (e.g., a micro-fluidic reactionchamber). Assembly reaction mixtures (e.g., liquid reaction samples) maybe transferred from one component of the system to another usingautomated devices and procedures (e.g., robotic manipulation and/ortransfer of samples and/or sample containers, including automatedpipetting devices, micro-systems, etc.). The system and any componentsthereof may be controlled by a control system.

Accordingly, method steps and/or aspects of the devices provided hereinmay be automated using, for example, a computer system (e.g., a computercontrolled system). A computer system on which aspects of the technologyprovided herein can be implemented may include a computer for any typeof processing (e.g., sequence analysis and/or automated device controlas described herein). However, it should be appreciated that certainprocessing steps may be provided by one or more of the automated devicesthat are part of the assembly system. In some embodiments, a computersystem may include two or more computers. For example, one computer maybe coupled, via a network, to a second computer. One computer mayperform sequence analysis. The second computer may control one or moreof the automated synthesis and assembly devices in the system. In otheraspects, additional computers may be included in the network to controlone or more of the analysis or processing acts. Each computer mayinclude a memory and processor. The computers can take any form, as theaspects of the technology provided herein are not limited to beingimplemented on any particular computer platform. Similarly, the networkcan take any form, including a private network or a public network(e.g., the Internet). Display devices can be associated with one or moreof the devices and computers. Alternatively, or in addition, a displaydevice may be located at a remote site and connected for displaying theoutput of an analysis in accordance with the technology provided herein.Connections between the different components of the system may be viawire, optical fiber, wireless transmission, satellite transmission, anyother suitable transmission, or any combination of two or more of theabove.

Each of the different aspects, embodiments, or acts of the technologyprovided herein can be independently automated and implemented in any ofnumerous ways. For example, each aspect, embodiment, or act can beindependently implemented using hardware, software or a combinationthereof. When implemented in software, the software code can be executedon any suitable processor or collection of processors, whether providedin a single computer or distributed among multiple computers. It shouldbe appreciated that any component or collection of components thatperform the functions described above can be generically considered asone or more controllers that control the above-discussed functions. Theone or more controllers can be implemented in numerous ways, such aswith dedicated hardware, or with general purpose hardware (e.g., one ormore processors) that is programmed using microcode or software toperform the functions recited above.

In this respect, it should be appreciated that one implementation of theembodiments of the technology provided herein comprises at least onecomputer-readable medium (e.g., a computer memory, a floppy disk, acompact disk, a tape, etc.) encoded with a computer program (i.e., aplurality of instructions), which, when executed on a processor,performs one or more of the above-discussed functions of the technologyprovided herein. The computer-readable medium can be transportable suchthat the program stored thereon can be loaded onto any computer systemresource to implement one or more functions of the technology providedherein. In addition, it should be appreciated that the reference to acomputer program which, when executed, performs the above-discussedfunctions, is not limited to an application program running on a hostcomputer. Rather, the term computer program is used herein in a genericsense to reference any type of computer code (e.g., software ormicrocode) that can be employed to program a processor to implement theabove-discussed aspects of the technology provided herein.

It should be appreciated that in accordance with several embodiments ofthe technology provided herein wherein processes are stored in acomputer readable medium, the computer implemented processes may, duringthe course of their execution, receive input manually (e.g., from auser).

Accordingly, overall system-level control of the assembly devices orcomponents described herein may be performed by a system controllerwhich may provide control signals to the associated nucleic acidsynthesizers, liquid handling devices, thermal cyclers, sequencingdevices, associated robotic components, as well as other suitablesystems for performing the desired input/output or other controlfunctions. Thus, the system controller along with any device controllerstogether form a controller that controls the operation of a nucleic acidassembly system. The controller may include a general purpose dataprocessing system, which can be a general purpose computer, or networkof general purpose computers, and other associated devices, includingcommunications devices, modems, and/or other circuitry or components toperform the desired input/output or other functions. The controller canalso be implemented, at least in part, as a single special purposeintegrated circuit (e.g., ASIC) or an array of ASICs, each having a mainor central processor section for overall, system-level control, andseparate sections dedicated to performing various different specificcomputations, functions and other processes under the control of thecentral processor section. The controller can also be implemented usinga plurality of separate dedicated programmable integrated or otherelectronic circuits or devices, e.g., hard wired electronic or logiccircuits such as discrete element circuits or programmable logicdevices. The controller can also include any other components ordevices, such as user input/output devices (monitors, displays,printers, a keyboard, a user pointing device, touch screen, or otheruser interface, etc.), data storage devices, drive motors, linkages,valve controllers, robotic devices, vacuum and other pumps, pressuresensors, detectors, power supplies, pulse sources, communication devicesor other electronic circuitry or components, and so on. The controlleralso may control operation of other portions of a system, such asautomated client order processing, quality control, packaging, shipping,billing, etc., to perform other suitable functions known in the art butnot described in detail herein.

Various aspects of the present invention may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

EXAMPLES Example 1 Mismatch Cleavage and Removal Using Surveyor™

An example of mismatch cleavage and removal is shown in FIGS. 2 and 3.With reference to FIG. 2, microarray spots 20 were subject to labelingwith in 0.005% Triton X-100, 10 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 7.5 mMdithiothreitol (DTT), 0.4 mM dATP, 0.4 mM dGTP, 0.4 mM dTTP, 4 μMCy3CdCTP, 0.4 uμM of universal primer, 0.04 U/μl of Klenow fragment DNApolymerase exo− at 37° C. for 60 minutes. In the control experiment(left panel), shuffled microarray 21 received no enzymatic treatment.The center panel shows shuffled microarray 22 that received 0.5 μlSurveyor™ per 20 μl reaction volume. The right panel is shuffledmicroarray 23 that received Surveyor™ per 20 ul reaction volume. 24, 25,and 16 are schematic representation of shuffled microarray 21, 22, and23, respectively.

The image of shuffled microarray 21 is the brightest, suggesting thatthe most amount of Cy3 dye remained on the shuffled microarray 21 than22 and 23. This is further substantiated by directly measuring the Cy3signal strength on shuffled microarray 21, 22, and 23. With reference toFIG. 3, reference numbers 31, 32, and 33 correspond to the average spotintensity of shuffled microarray 21, 22, and 23, respectively.Therefore, Surveyor™ treatment effectively removed mismatch-containingduplexes, resulting in lesser amount of Cy3 signals.

Example 2 Production of High Fidelity Oligonucleotides Using Surveyor™Cleavage

Exemplary molecular reaction flow, process flow, and reagent flow forproducing high fidelity oligonucleotides using Surveyor™ cleavage areshown in FIG. 4. Seven (7) steps were used in this example.

Step 1: Chip Prehybridization. Microarray was prehybridized with 0.005%Triton X-100, 0.2 mg/ml acytylated Bovine Serum Albumin, 10 mM Tris-HCl(pH 7.4), 5 mM MgCl2, 7.5 mM dithiothreitol (DTT) at 37° C. for 30minutes.

Step 2: Primer Extension. Complementary strands on the chip weresynthesized in 0.005% Triton X-100, 10 mM Tris-HCl (pH 7.4), 5 mM MgCl2,7.5 mM dithiothreitol (DTT), 0.4 mM dNTPs, 0.4 μM of universal primer,0.04 U/μl of Klenow fragment DNA polymerase exo− at 37° C. for 60minutes.

Step 3: Surveyor™ hybe wash. Unincorporated nucleotides were removedwith 0.9M NaCl, 60 mM NaH2PO4 and 0.005% Triton X-100. Chip was washedin Surveyor™ hybe buffer three times at room temperature.

Step 4: Surveyor™ reaction wash. Chip was washed in Surveyor™ reactionbuffer three times at room temperature. Composition of Surveyor™reaction buffer: 20 mM Tris-HCl (pH 7.4), 10 mM MgCl2 and 25 mM KCl.

Step 5: Shuffling. The chip was heated to 94° C. in a Surveyor™ reactionbuffer for 3 minutes for DNA melting followed by room temperatureincubation for 60 minutes for heteroduplex formation.

Step 6: Surveyor™ treatment. The Surveyor™ reaction buffer from step 5was removed and the chip was treated with Surveyor™/enhancer in 20 mMTris-HCl (pH 7.4), 10 mM MgCl2 and 25 mM KCl and incubated at 42° C. for20 minutes. Two different concentrations of Surveyor™/enhancer wereused: 1). 0.5 μl/20 μl reaction and 1.5 μl/20 μl reaction. Surveyor™ andenhancer were used in equal volumes.

Step 7: Surveyor™ after reaction wash. Chip was washed with 0.9M NaCl,60 mM NaH2PO4, 0.005% Triton X-100 and 6 mM EDTA for three times at roomtemperature to remove cleaved error-prone heteroduplexes.

EQUIVALENTS

The present invention provides among other things novel methods anddevices for high-fidelity gene synthesis and assembly. While specificembodiments of the subject invention have been discussed, the abovespecification is illustrative and not restrictive. Many variations ofthe invention will become apparent to those skilled in the art uponreview of this specification. The full scope of the invention should bedetermined by reference to the claims, along with their full scope ofequivalents, and the specification, along with such variations.

INCORPORATION BY REFERENCE

Reference is made to PCT application PCT/US09/55267, now InternationalPublication No. WO2010/025310; to PCT application PCT/US2010/055298, nowInternational Publication No. WO2011/056872; and to U.S. Provisionalapplication 61/264,632 filed on Nov. 25, 2009. All publications, patentsand sequence database entries mentioned herein are hereby incorporatedby reference in their entirety as if each individual publication orpatent was specifically and individually indicated to be incorporated byreference.

1. A method for producing a population of double-strandedoligonucleotides having improved fidelity on a solid support, the methodcomprising: (a) synthesizing a first plurality of oligonucleotides in achain extension reaction using a second plurality of support-boundoligonucleotides as templates, wherein the second plurality ofoligonucleotides are bound on a solid support at their 3′ ends andcomprise an error-containing oligonucleotide having a sequence error atan error-containing position, thereby producing a first plurality ofduplexes, wherein the first plurality of duplexes compriseshomoduplexes; (b) denaturing the first plurality of duplexes, therebyreleasing the first plurality of oligonucleotides, wherein the releasedfirst plurality of oligonucleotides comprise error-free oligonucleotidesthat are free of error at a position corresponding to theerror-containing position of the error-containing oligonucleotide in thesecond plurality of oligonucleotides; (c) contacting the released firstplurality of oligonucleotides with the second plurality ofoligonucleotides under hybridization conditions to form a secondplurality of duplexes, wherein the second plurality of duplexes compriseone or more mismatch-containing heteroduplex formed between theerror-containing oligonucleotide and one of the error-freeoligonucleotides; (d) cleaving the mismatch-containing heteroduplex by amismatch recognizing and cleaving component; and (e) removing themismatch-containing heteroduplex, thereby producing the population ofdouble-stranded oligonucleotides having improved fidelity on the solidsupport.
 2. The method of claim 1 further comprising selectivelydenaturing the population of double-stranded oligonucleotides havingimproved fidelity.
 3. A method of assembling nucleic acid polymerscomprising the steps of: (a) producing two or more populations ofdouble-stranded oligonucleotides having improved fidelity according tothe method of claim 1; (b) denaturing selected populations of thedouble-stranded oligonucleotides from the two or more populations ofdouble-stranded oligonucleotides having improved fidelity, therebyreleasing at least a first desirable pool and a second desirable pool ofsingle-stranded oligonucleotides having improved fidelity in a solution;(c) combining the at least a first desirable pool and a second desirablepool of single-stranded oligonucleotides; (d) subjecting thesingle-stranded oligonucleotides to conditions suitable forhybridization, and (e) assembling the nucleic acid polymers by ligation,by chain extension, or by chain extension and ligation of thesingle-stranded oligonucleotides.
 4. The method of claim 1 wherein thesecond plurality of oligonucleotides are chemically synthesized on thesolid support and immobilized within one or more features on the solidsupport.
 5. The method of claim 1 wherein the first plurality ofoligonucleotides are enzymatically synthesized on the solid support. 6.The method of claim 1 wherein the second plurality of oligonucleotidesare attached to two or more features on the solid support and whereinafter step (b), one or more of the first plurality of oligonucleotidesdiffuse away from the two or more features.
 7. The method of claim 1wherein the mismatch recognizing and cleaving component comprises amismatch endonuclease.
 8. The method of claim 1 wherein the mismatchrecognizing and cleaving component performs a chemical cleavage.
 9. Themethod of claim 1 wherein the removing step comprises buffer exchange.10. The method of claim 1 wherein the solid support is a microarray. 11.A method for producing at least one support-bound error-freeoligonucleotide having a predefined sequence on a solid support, themethod comprising: (a) synthesizing a first plurality ofoligonucleotides on a solid support using a second plurality ofsupport-bound oligonucleotides as templates in the presence of at leastone primer, wherein the second plurality of support-boundoligonucleotides is bound on the solid support at their 3′ ends, whereinthe at least one primer is complementary to a primer binding site on thesecond plurality of oligonucleotides, wherein each of the secondplurality of oligonucleotides has a predefined sequence, and wherein atleast one of the second plurality of support-bound oligonucleotidescomprises a sequence error; (b) releasing the first plurality ofoligonucleotides; (c) contacting the second plurality of support-boundoligonucleotides with the first plurality of oligonucleotides underhybridization conditions to form a plurality of double-strandedoligonucleotides, wherein the plurality of double-strandedoligonucleotides comprises a double-stranded oligonucleotide having amismatch with the sequence error; (d) contacting and cleaving the secondplurality of double-stranded oligonucleotides with a mismatch bindingagent, wherein the mismatch binding agent selectively binds and cleavesthe double-stranded oligonucleotide having the mismatch; and (e)removing the double-stranded oligonucleotide having the mismatch,thereby producing the at least one support-bound error-freeoligonucleotide having the predefined sequence on the solid support. 12.The method of claim 11 wherein the mismatch binding agent is a mismatchspecific endonuclease.
 13. The method of claim 12 wherein the mismatchspecific endonuclease cleaves the nucleotide at the region of themismatch.
 14. The method of claim 12 wherein the mismatch specificendonuclease is a CEL enzyme.
 15. The method of claim 11 wherein thefirst plurality of oligonucleotides in the releasing step is releasedunder denaturing conditions.
 16. The method of claim 11 wherein thesecond plurality of oligonucleotides is bound to one or more discretefeatures of the solid support and wherein the one or more features areselectively hydrated such that the second plurality of oligonucleotidesare present within one or more droplets.
 17. The method of claim 16wherein the synthesizing step further comprises selectively hydratingthe one or more features by spotting a solution comprising the at leastone primer, a polymerase, dNTPs, and a buffer capable of promotingprimer extension.
 18. The method of claim 11 further comprisingreleasing at least one error-free single-stranded oligonucleotide insolution.
 19. A method for producing high fidelity oligonucleotides on asolid support, the method comprising: (a) contacting, on a solidsupport, a plurality of support-bound single-stranded oligonucleotideswith a solution comprising a primer, nucleotides and a polymerase enzymeunder conditions suitable for a template-dependent synthesis reaction,wherein the plurality of support-bound single-stranded oligonucleotidesare bound on the solid support at the 3′ end and comprising error-freeoligonucleotides and error-containing oligonucleotides, therebyproducing a plurality of double-stranded oligonucleotides comprisingsynthesized complementary oligonucleotides base paired with thesupport-bound single-stranded oligonucleotides; (b) denaturing theplurality of double-stranded oligonucleotides such that the synthesizedcomplementary oligonucleotides are released into a solution; (c)reannealing the synthesized complementary oligonucleotides to thesupport-bound single-stranded oligonucleotides, thereby to producereannealed double-stranded oligonucleotides comprising homoduplexes andheteroduplexes, wherein the heteroduplexes each comprise a mismatch; (d)exposing the reannealed double-stranded oligonucleotides to a mismatchrecognizing and cleaving component under conditions suitable forcleavage of the heteroduplexes, thereby cleaving at least a portion ofthe heteroduplexes; (e) removing at least a portion of the cleavedheteroduplexes, thereby producing a population of error-freesupport-bound double-stranded oligonucleotides and a population oftruncated duplexes; and (f) selectively denaturing the population oferror-free support-bound double-stranded oligonucleotides, therebyproducing single-stranded high fidelity oligonucleotides.